1. Microbiology and Infectious Disease
Download icon

Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals

  1. Andrew A Bridges
  2. Bonnie L Bassler  Is a corresponding author
  1. Department of Molecular Biology, Princeton University, United States
  2. The Howard Hughes Medical Institute, United States
Research Article
  • Cited 1
  • Views 1,093
  • Annotations
Cite this article as: eLife 2021;10:e65487 doi: 10.7554/eLife.65487

Abstract

The global pathogen Vibrio cholerae undergoes cycles of biofilm formation and dispersal in the environment and the human host. Little is understood about biofilm dispersal. Here, we show that MbaA, a periplasmic polyamine sensor, and PotD1, a polyamine importer, regulate V. cholerae biofilm dispersal. Spermidine, a commonly produced polyamine, drives V. cholerae dispersal, whereas norspermidine, an uncommon polyamine produced by vibrios, inhibits dispersal. Spermidine and norspermidine differ by one methylene group. Both polyamines control dispersal via MbaA detection in the periplasm and subsequent signal relay. Our results suggest that dispersal fails in the absence of PotD1 because endogenously produced norspermidine is not reimported, periplasmic norspermidine accumulates, and it stimulates MbaA signaling. These results suggest that V. cholerae uses MbaA to monitor environmental polyamines, blends of which potentially provide information about numbers of ‘self’ and ‘other’. This information is used to dictate whether or not to disperse from biofilms.

Introduction

Bacteria frequently colonize environmental habitats and infection sites by forming surface-attached multicellular communities called biofilms. Participating in the biofilm lifestyle allows bacteria to collectively acquire nutrients and resist threats (Flemming et al., 2016). By contrast, the individual free-swimming state allows bacteria to roam. The global pathogen Vibrio cholerae undergoes repeated rounds of clonal biofilm formation and disassembly, and both biofilm formation and biofilm exit are central to disease transmission as V. cholerae alternates between the marine niche and the human host (Conner et al., 2016; Gallego-Hernandez et al., 2020; Tamayo et al., 2010). The V. cholerae biofilm lifecycle occurs in three stages. First, a founder cell attaches to a substate; second, through cycles of growth, division, and extracellular matrix secretion, the biofilm matures; and finally, when the appropriate environmental conditions are detected, cells leave the biofilm through a process termed dispersal (Bridges et al., 2020). Bacterial cells liberated through dispersal can depart their current environment and repeat the lifecycle in a new niche, such as in a new host (Guilhen et al., 2017). In general, the initial stages of the biofilm lifecycle, from attachment through maturation, have been well studied in V. cholerae and other bacterial species, whereas the dispersal phase remains underexplored. Components underlying biofilm dispersal are beginning to be revealed. For example, in many bacterial species, the Lap system that is responsible for cell attachments during biofilm formation is also involved in dispersal (Boyd et al., 2012; Christensen et al., 2020; Gjermansen et al., 2005; Kitts et al., 2019; Newell et al., 2011).

To uncover mechanisms controlling V. cholerae biofilm dispersal, we recently developed a brightfield microscopy assay that allows us to follow the entire biofilm lifecycle (Bridges and Bassler, 2019). We combined this assay with mutagenesis and high-content imaging to identify mutants that failed to properly disperse (Bridges et al., 2020). Our screen revealed genes encoding proteins that fall into three functional groups: signal transduction, matrix degradation, and cell motility. Our hypothesis is that these classes of components act in sequence to drive biofilm dispersal. First, the cues that induce dispersal are detected by the signal transduction proteins. Second, activation of matrix digestion components occurs. Finally, cell motility engages and permits cells to swim away from the disassembling biofilm. The majority of the genes identified in the screen encoded signal transduction proteins. Elsewhere, we characterized one of the signaling systems identified in the screen, a new two-component phospho-relay that we named DbfS-DbfR (for Dispersal of Biofilm Sensor and Dispersal of Biofilm Regulator) that controls biofilm dispersal (Bridges et al., 2020). Of the remaining signaling proteins identified in the screen, four are proteins involved in regulating production/degradation of, or responding to, the second messenger molecule cyclic diguanylate (c-di-GMP).

c-di-GMP regulates biofilm formation in many bacteria including V. cholerae in which low c-di-GMP levels correlate with motility and high c-di-GMP levels promote surface attachment and matrix production, and thus, biofilm formation (Conner et al., 2017). c-di-GMP is synthesized by enzymes that contain catalytic GGDEF domains that have diguanylate cyclase activity (Conner et al., 2017). c-di-GMP is degraded by proteins with EAL or HD-GYP domains that possess phosphodiesterase activity. V. cholerae encodes >50 proteins harboring one or both of these domains, underscoring the global nature of c-di-GMP signaling in this pathogen. The activities of these enzymes are often regulated by environmental stimuli through ligand binding sensory domains; however, in most cases, the identities of ligands for GGDEF- and EAL-containing enzymes are unknown (Römling et al., 2013). Of the c-di-GMP regulatory proteins identified in our dispersal screen, only one, the hybrid GGDEF/EAL protein, MbaA, which has previously been shown to regulate V. cholerae biofilm formation (Bomchil et al., 2003), has a defined environmental stimulus: MbaA responds to the ubiquitous family of small molecules called polyamines, aliphatic cations containing two or more amine groups (Miller-Fleming et al., 2015). Our dispersal screen also identified PotD1, a periplasmic binding protein that mediates polyamine import and that has also been shown to control V. cholerae biofilm formation (Cockerell et al., 2014; McGinnis et al., 2009). Thus, identification of these two genes in our screen suggested that beyond roles in biofilm formation, polyamine detection could be central to biofilm dispersal. In the present work, we explore this new possibility.

Polyamines are essential small molecules derived from amino acids and were likely produced by the last universal common ancestor (Michael, 2018; Miller-Fleming et al., 2015). Different organisms make distinct blends of polyamines (Michael, 2018), and they are involved in biological processes ranging from stress responses to signal transduction to protein synthesis to siderophore production (Karatan et al., 2005; McGinnis et al., 2009; Michael, 2018). In some cases, mechanisms underlying polyamine function are known. For example, the eukaryotic translation initiation factor 5A is modified with hypusine that is derived from the polyamine spermidine (Puleston et al., 2019). In most cases, however, the mechanisms by which polyamines control physiology remain mysterious (Miller-Fleming et al., 2015).

Polyamines are known to be involved in biofilm development in many bacterial species (Hobley et al., 2014; Karatan and Michael, 2013; Michael, 2018; Nesse et al., 2015; Patel et al., 2006). Intriguingly, the biofilm alterations that occur in response to a given polyamine vary among species. In V. cholerae, norspermidine, a rare polyamine produced by Vibrionaceae and select other organisms, promotes biofilm formation (Karatan et al., 2005; Lee et al., 2009). In contrast, the nearly ubiquitous polyamine spermidine, which differs from norspermidine only by a single methylene group, is not produced at substantial levels by V. cholerae and it represses V. cholerae biofilm formation (Figure 1Karatan et al., 2005; Lee et al., 2009; McGinnis et al., 2009). Likewise, the polyamine spermine, typically made by eukaryotes, also represses V. cholerae biofilm formation (Sobe et al., 2017). Both spermidine and spermine are abundant in the human intestine (Benamouzig et al., 1997; McEvoy and Hartley, 1975; Osborne and Seidel, 1990). These results have led to the hypothesis that V. cholerae detects norspermidine as a measure of ‘self’ and spermidine and spermine as measures of ‘other’ to assess the species composition of the vicinal community (Sobe et al., 2017; Wotanis et al., 2017). V. cholerae funnels that information internally to determine whether or not to make a biofilm (Sobe et al., 2017; Wotanis et al., 2017). V. cholerae produces intracellular norspermidine; however, norspermidine has not been detected in cell-free culture fluids of laboratory-grown strains (Parker et al., 2012). Thus, if norspermidine does indeed enable V. cholerae to take a census of ‘self,’ it is apparently not via a canonical quorum-sensing-type mechanism.

Polyamine sensing in V. cholerae.

Schematic showing the previously proposed polyamine detection and import mechanisms in V. cholerae. Norspermidine (Nspd, triangles) promotes biofilm formation and spermidine (Spd, circles) represses biofilm formation. The primarily eukaryotic polyamine spermine (not pictured) also signals through the NspS-MbaA pathway. See text for details. OM: outer membrane; IM: inner membrane.

As mentioned above, information contained in extracellular polyamines is transduced internally by MbaA, which is embedded in the inner membrane. MbaA contains a periplasmic domain that interacts with the periplasmic binding protein NspS. nspS is located immediately upstream of mbaA in the chromosome (Cockerell et al., 2014; Karatan et al., 2005; Young et al., 2021). MbaA also possesses cytoplasmic GGDEF (SGDEF in MbaA) and EAL (EVL in MbaA) domains (Figure 1). Genetic evidence suggests that when NspS is bound to norspermidine, the complex associates with MbaA and biofilm formation is promoted (Cockerell et al., 2014). Specifically, NspS interaction with MbaA inhibits MbaA phosphodiesterase activity, leading to increased c-di-GMP levels, and in turn, elevated biofilm formation (Figure 1, left panel) (Cockerell et al., 2014). It is proposed that Apo-NspS and NspS bound to spermidine or spermine do not bind to MbaA, and thus, under this condition, biofilm formation is reduced (Cockerell et al., 2014; Sobe et al., 2017). In this case, MbaA phosphodiesterase activity is not inhibited, which leads to decreased c-di-GMP levels and repression of biofilm formation (Figure 1, right panel). Currently, it is not known whether or not MbaA has diguanylate cyclase activity. The purified MbaA cytoplasmic domain functions as a phosphodiesterase in vitro (Cockerell et al., 2014).

In a proposed second regulatory mechanism, norspermidine and spermidine are thought to control biofilm formation via import through the inner membrane ABC transporter, PotABCD1 (McGinnis et al., 2009). Following internalization, via an undefined cytoplasmic mechanism, polyamines are proposed to modulate biofilm formation (Figure 1McGinnis et al., 2009). This hypothesis was based on the finding that elimination of PotD1, which is required for import of norspermidine and spermidine, resulted in elevated biofilm formation. Importantly, the studies reporting the MbaA and PotD1 findings used quorum-sensing-deficient V. cholerae strains (Joelsson et al., 2006) that likely cannot disperse from biofilms. Furthermore, the end-point biofilm assays used could not differentiate between enhanced biofilm formation and the failure to disperse.

Here, we combine real-time biofilm lifecycle measurements, mutagenesis, a reporter of cytoplasmic c-di-GMP levels, and measurements of intra- and extracellular polyamine concentrations to define the roles that norspermidine and spermidine play in controlling the V. cholerae biofilm lifecycle. In wildtype V. cholerae, exogenous norspermidine and spermidine inversely alter cytoplasmic c-di-GMP levels and they exert their effects through the NspS-MbaA circuit. Norspermidine promotes biofilm formation and suppresses biofilm dispersal. Spermidine represses biofilm formation and promotes biofilm dispersal. Both the MbaA SGDEF and EVL domains are required for polyamine control of the biofilm lifecycle. We provide evidence that MbaA synthesizes c-di-GMP in the presence of norspermidine and degrades c-di-GMP in the presence of spermidine and that is what drives V. cholerae to form and disperse from biofilms, respectively. When MbaA is absent, V. cholerae is unable to alter the biofilm lifecycle in response to extracellular polyamines. We demonstrate that polyamine internalization via PotD1 is not required to promote V. cholerae entrance or exit from biofilms, but rather, periplasmic detection of polyamines by MbaA is the key regulatory step. Specifically, our results suggest that the ΔpotD1 mutant fails to disperse because it is unable to reimport self-secreted norspermidine. The consequence is that excess periplasmic norspermidine accumulates, is detected by MbaA, and leads to production of c-di-GMP and suppression of biofilm dispersal. Collectively, our work reveals the mechanisms by which V. cholerae detects and transduces the information contained in polyamine signals into modulation of its biofilm lifecycle. We propose that the polyamine sensing mechanisms revealed in this study allow V. cholerae to distinguish relatives from competitors and potentially the presence of predators in the vicinity and, in response, modify its biofilm lifecycle to appropriately colonize territory or disperse from an existing community.

Results

The polyamine signaling proteins MbaA and PotD1 regulate biofilm dispersal by changing c-di-GMP levels

Our combined mutagenesis-imaging screen identified the inner membrane polyamine sensor, MbaA, and the periplasmic polyamine binding protein PotD1 as essential for proper V. cholerae biofilm dispersal, motivating us to explore the mechanisms underlying these effects. To probe polyamine signaling across the full biofilm lifecycle, we used our established brightfield imaging assay. In the case of WT V. cholerae, peak biofilm biomass is reached at ~8–9 hr of growth, and subsequently, dispersal occurs, and is completed by ~12 hr post inoculation (Figure 2A, B). Deletion of mbaA caused a mild biofilm dispersal defect with no detectable difference in peak biofilm biomass compared to WT, a less than 1 hr delay in the onset of dispersal, and 27% biomass remaining at 16 hr (Figure 2A, B). Expression of mbaA from an ectopic locus in the ΔmbaA mutant complemented the biofilm dispersal defect (Figure 2—figure supplement 1A). Thus, MbaA is required for WT V. cholerae biofilm dispersal. The ΔpotD1 mutant exhibited a 60% greater peak biofilm biomass than WT and nearly all of the biomass remained at 16 hr, indicating that PotD1 both represses biofilm formation and promotes biofilm dispersal (Figure 2A, B). Introduction of potD1 at an ectopic locus in the ΔpotD1 mutant drove premature biofilm dispersal and reduced overall biofilm formation (Figure 2—figure supplement 1B). The differences in severity between the dispersal phenotypes of the ΔmbaA and ΔpotD1 mutants are noteworthy because these strains behaved similarly in the previous end-point biofilm formation assays (McGinnis et al., 2009).

Figure 2 with 2 supplements see all
Polyamine signaling regulates V. cholerae biofilm dispersal.

(A) Representative images of the designated V. cholerae strains at 16 hr. (B) Quantitation of biofilm biomass over time measured by time-lapse microscopy for WT V. cholerae and the designated mutants. In all cases, N = 3 biological and N = 3 technical replicates, ± SD (shaded). a.u.: arbitrary unit. (C) Relative c-di-GMP reporter signals for the indicated strains. Values are expressed as the percentage difference relative to the WT strain. N = 4 biological replicates. Each black bar shows the sample mean. Unpaired t-tests were performed for statistical analysis, with p values denoted as *p<0.05; ***p<0.001. (D) The corresponding PvpsL-lux outputs for the strains and growth conditions in (B). For vpsL-lux measurements, N = 3 biological replicates, ± SD (shaded). RLU: relative light units.

MbaA has previously been shown to possess phosphodiesterase activity in vitro (Cockerell et al., 2014), thus, we reasoned that MbaA functions by altering cytoplasmic c-di-GMP levels, the consequence of which is changes in expression of genes encoding biofilm components. As far as we are aware, no connection has yet been made between PotD1 and cytoplasmic c-di-GMP levels. To determine if the observed biofilm dispersal defects in the ΔmbaA and ΔpotD1 mutants track with altered c-di-GMP levels, we compared the relative cytoplasmic c-di-GMP levels in the WT, ΔmbaA, and ΔpotD1 strains. To do this, we employed a fluorescent reporter construct in which expression of turboRFP is controlled by two c-di-GMP-responsive riboswitches (Zhou et al., 2016). The TurboRFP signal is normalized to constitutively produced AmCyan encoded on the same plasmid. Previous studies have demonstrated a linear relationship between reporter output and c-di-GMP levels measured by mass spectrometry (Zhou et al., 2016). Indeed, the reporter showed that deletion of mbaA caused only a moderate increase in cytoplasmic c-di-GMP (8% higher than WT), while the ΔpotD1 mutant produced 39% more signal than WT (Figure 2C). Thus, MbaA, as expected, mediates changes in c-di-GMP levels, and moreover PotD1-mediated import of polyamines also influences cytoplasmic c-di-GMP concentrations.

In V. cholerae, increased cytoplasmic c-di-GMP levels are associated with elevated extracellular matrix production (called VPS for vibrio polysaccharide) and, in turn, increased biofilm formation. Using a PvpsL-lux promoter fusion that reports on the major matrix biosynthetic operon, we previously showed that matrix gene expression decreases as cells transition from the biofilm to the planktonic state, suggesting that repression of matrix production genes correlates with biofilm dispersal. We wondered how the increased c-di-GMP levels present in the ΔmbaA and ΔpotD1 mutants impinged on vpsL expression. Using the vpsL-lux reporter, we found that the light production patterns mirrored the severities of the dispersal phenotypes and the magnitudes of changes in cytoplasmic c-di-GMP levels: the ΔmbaA mutant had a light production profile similar to WT, while the ΔpotD1 mutant produced 10-fold more light than WT throughout growth (Figure 2D). These results indicate that the ΔmbaA mutant makes normal levels and the ΔpotD1 mutant produces excess matrix.

As mentioned in the Introduction, our screen revealed three additional genes in c-di-GMP signaling pathways involved in V. cholerae biofilm dispersal. While not the focus of the current work, we performed preliminary characterization by deleting these genes and making measurements of the mutants' biofilm lifecycle phenotypes, assessing them for changes in cytoplasmic c-di-GMP levels, and quantifying their matrix production profiles (Figure 2—figure supplement 2A–D).

Norspermidine and spermidine inversely regulate V. cholerae biofilm dispersal

Above, we show that polyamine signaling proteins are required for normal V. cholerae biofilm dispersal. Previous studies demonstrated that norspermidine and spermidine have opposing effects on biofilm formation in end-point assays (Karatan et al., 2005; McGinnis et al., 2009). We wondered if and how these two polyamines affect each stage of the biofilm lifecycle – biofilm formation and biofilm dispersal. To investigate their roles, we assayed the biofilm lifecycle in WT V. cholerae and our mutants following exogenous administration of norspermidine and spermidine, alone and in combination. Addition of 100 µM norspermidine strongly promoted biofilm formation and completely prevented biofilm dispersal while 100 µM spermidine dramatically reduced biofilm formation and promoted premature biofilm dispersal (Figure 3A, Video 1). vpsL-lux expression increased by >10-fold following norspermidine treatment and decreased >10-fold following spermidine treatment (Figure 3B). These results show that norspermidine and spermidine have opposing activities with regard to biofilm formation and dispersal: norspermidine drives biofilm formation by inducing matrix production, and this prevents biofilm dispersal and spermidine, by suppressing matrix production prevents biofilm formation and drives biofilm dispersal.

Figure 3 with 1 supplement see all
Periplasmic detection of polyamines controls V. cholerae biofilm dispersal.

(A) Quantitation of biofilm biomass over time measured by time-lapse microscopy following addition of water (Ctrl), 100 µM norspermidine, or 100 µM spermidine to WT V. cholerae. (B) Light output from the PvpsL-lux reporter for the treatments in (A) over the growth curve. (C) c-di-GMP reporter output at the indicated polyamine concentrations for WT V. cholerae. Relative reporter signal (% difference) is displayed as a heatmap (teal and purple represent the lowest and highest reporter output, respectively). (D) As in (A) for the ΔmbaA mutant. (E) As in (C) for the ΔmbaA mutant. (F) As in (C) for the ΔnspS mutant. Biofilm biomass data are represented as means normalized to the peak biofilm biomass of the Ctrl condition. In all biofilm biomass measurements, N = 3 biological and N = 3 technical replicates, ± SD (shaded). a.u.: arbitrary unit. In vpsL-lux measurements, N = 3 biological replicates, ± SD (shaded). RLU: relative light units. For the c-di-GMP reporter assays, values are expressed as the percentage difference relative to the untreated WT strain, allowing comparisons to be made across all heatmaps in all figures in this article. The same color bar applies to all heatmaps in this article. For each condition, N = 3 biological replicates. Numerical values and associated SDs are available in Supplementary file 1.

Video 1
Representative time-lapse images of the biofilm lifecycles of the WT, ΔmbaA, ΔpotD1, and ΔmbaA ΔpotD1 V. cholerae strains following treatment with water (Ctrl), 100 µM norspermidine (Nspd), or 100 µM spermidine (Spd).

We wondered by what mechanism the information encoded in exogenous polyamines was translated into alterations in matrix gene expression and subsequent changes in biofilm dispersal. We reasoned that MbaA and/or PotD1-mediated changes in cytoplasmic c-di-GMP levels could be responsible. To test this possibility, we assessed how changes in cytoplasmic c-di-GMP levels tracked with changes in extracellular polyamine levels by measuring the c-di-GMP reporter output in response to supplied mixtures of norspermidine and spermidine (Figure 3C). In the heatmaps, teal and purple represent the lowest and highest values of reporter output, respectively. When provided alone and above a concentration of 10 µM, norspermidine and spermidine strongly increased and decreased, respectively, c-di-GMP reporter activity. At the limit, relative to the WT, norspermidine increased the c-di-GMP reporter output by ~60% while spermidine reduced reporter output by ~25%. Notably, consistent with previous end-point biofilm assays, when norspermidine was present above 50 µM, it overrode the effect of exogenous addition of spermidine, irrespective of spermidine concentration (Figure 3CMcGinnis et al., 2009). Thus, V. cholerae cytoplasmic c-di-GMP levels are responsive to extracellular blends of polyamines. When norspermidine is abundant, V. cholerae produces high levels of c-di-GMP, matrix expression is increased, and biofilms do not disperse. In contrast, when spermidine is abundant and norspermidine is absent or present below a threshold concentration, V. cholerae c-di-GMP levels drop, matrix production is repressed, and biofilm dispersal occurs.

MbaA transduces external polyamine information internally to control biofilm dispersal

Our data show that both the MbaA periplasmic polyamine sensor and the PotD1 polyamine importer are required for biofilm dispersal. Our next goal was to distinguish the contribution of periplasmic detection from cytoplasmic import of polyamines to the biofilm lifecycle. We began with periplasmic polyamine detection, mediated by NspS together with MbaA. We supplied norspermidine or spermidine to the ΔmbaA mutant and monitored biofilm biomass over time. To our surprise, the ΔmbaA mutant was impervious to the addition of either polyamine as both polyamines caused only a very modest reduction in overall biofilm biomass, and dispersal timing resembled the untreated ΔmbaA control (Figure 3D, Video 1). Consistent with this result, titration of the polyamines alone and in combination onto the ΔmbaA strain carrying the c-di-GMP reporter did not substantially alter reporter output (Figure 3E). These results show that the dynamic response of WT to polyamines requires MbaA. We suspect that the minor reduction in biofilm production that occurred in the ΔmbaA mutant when supplied polyamines is due to non-specific effects, perhaps via interaction of polyamines with negatively charged matrix components. We next investigated the role of the polyamine periplasmic binding protein NspS that transmits polyamine information to MbaA. In the absence of its partner polyamine binding protein, NspS, MbaA is thought to function as a constitutive phosphodiesterase (Cockerell et al., 2014). Consistent with this model, deletion of nspS in an otherwise WT strain reduced overall peak biofilm biomass by 65% and dispersal initiated 4 hr prior to when dispersal begins in the WT (Figure 3—figure supplement 1). In the ΔnspS mutant, c-di-GMP levels were lower than in the WT as judged by the c-di-GMP reporter (Figure 3F), showing that MbaA is locked as a constitutive phosphodiesterase. Exogenous addition of polyamines had no effect on c-di-GMP levels (Figure 3F). Together, these findings demonstrate that the WT V. cholerae response to polyamines is controlled by the NspS-MbaA polyamine sensing circuit.

Both the MbaA EVL and SGDEF domains are required for MbaA to detect polyamines

We wondered how the putative MbaA phosphodiesterase and diguanylate cyclase enzymatic activities contribute to the V. cholerae responses to norspermidine and spermidine. The cytoplasmic domain of MbaA exhibits phosphodiesterase but not diguanylate cyclase activity in vitro (Cockerell et al., 2014). We reasoned that, when an intact regulatory domain is present, MbaA could possess both enzymatic activities, with the activity of each domain inversely controlled by NspS binding. To probe the role of each domain, we introduced inactivating point mutations in the catalytic motifs. To ensure that our mutations did not destabilize MbaA, we first fused MbaA to 3xFLAG and introduced the gene encoding the fusion onto the chromosome at the mbaA locus. Tagging did not alter MbaA control over the biofilm lifecycle (Figure 4—figure supplement 1A, Video 2). To inactivate the MbaA phosphodiesterase activity, we substituted the conserved catalytic residue E553 with alanine, converting EVL to AVL (referred to as EVL* henceforth). This change did not alter MbaA-3xFLAG abundance (Figure 4—figure supplement 1B). V. cholerae harboring MbaAEVL* exhibited an increase in biofilm biomass and a strong biofilm dispersal defect (Figure 4A), and only a modest response to exogenous polyamines, with norspermidine eliciting some inhibition of biofilm dispersal and spermidine driving a small reduction in overall biofilm biomass (Figure 4B, Video 2). Treatment with norspermidine did increase c-di-GMP levels in V. cholerae carrying MbaAEVL* as judged by the reporter output (Figure 4C), albeit not to the level of WT (Figure 3C), suggesting that the V. cholerae mbaAEVL* mutant, which is incapable of c-di-GMP degradation, retains the capacity to synthesize some c-di-GMP via the MbaA SGDEF domain. In contrast, the V. cholerae mbaAEVL* strain displayed little reduction in c-di-GMP levels in response to spermidine, presumably because it lacks the phosphodiesterase activity required to degrade c-di-GMP (Figure 4C). Thus, despite the fact that the purified cytoplasmic domain functioned only as a phosphodiesterase in vitro, our results suggest that, in vivo, MbaA is capable of synthesizing c-di-GMP in the presence of norspermidine. To validate this prediction, we altered the conserved catalytic residues D426 and E427 to alanine residues, yielding the inactive MbaA SGAAF variant (referred to as SGDEF*). These substitutions did not affect protein levels (Figure 4—figure supplement 1B). In every regard, the mbaASGDEF* mutant behaved identically to the ΔmbaA mutant. The V. cholerae mbaASGDEF* mutant exhibited a modest biofilm dispersal defect (Figure 4A) and biofilm biomass was reduced in response to exogenous norspermidine and spermidine (Figure 4D, Video 2). Furthermore, addition of polyamines to the mbaASGDEF* mutant harboring the c-di-GMP reporter did not drive substantial changes in reporter output (Figure 4E). These results show that the MbaA SGDEF domain is indispensable for the MbaA response to polyamines.

Figure 4 with 1 supplement see all
Both the MbaA SGDEF and EVL domains are required for regulation of V. cholerae biofilm dispersal.

(A) Quantitation of biofilm biomass over time for the V. cholerae strains carrying mbaA-3xFLAG, mbaAEVL*−3xFLAG, and mbaASGDEF*−3xFLAG. (B) Quantitation of biofilm biomass over time measured by time-lapse microscopy for V. cholerae carrying mbaAEVL*−3xFLAG following addition of water (Ctrl), 100 µM norspermidine, or 100 µM spermidine. (C) c-di-GMP reporter output at the indicated polyamine concentrations for V. cholerae carrying mbaAEVL*−3xFLAG. Relative reporter signal (% difference) is displayed as a heatmap (teal and purple represent the lowest and highest reporter output, respectively). (D) As in (B) for the mbaASGDEF*−3xFLAG mutant. (E) As in (C) for the mbaASGDEF*−3xFLAG mutant. (F) Schematic representing the proposed MbaA activities in response to norspermidine and spermidine. Biofilm biomass data are represented as means normalized to the peak biofilm biomass of the WT strain or Ctrl condition. In all cases, N = 3 biological and N = 3 technical replicates, ± SD (shaded). a.u.: arbitrary unit. In the c-di-GMP reporter assays, values are expressed as the percentage difference relative to the untreated WT strain, allowing comparisons to be made across all heatmaps in all figures in this article. The same color bar applies to all panels in this article. For each condition, N = 3 biological replicates. Numerical values and associated SDs are available in Supplementary file 1. OM: outer membrane; IM: inner membrane.

Video 2
Representative time-lapse images of the biofilm lifecycles of the mbaA-3xFLAG, mbaAEVL*−3xFLAG, and mbaASGDEF*−3xFLAG V. cholerae strains following treatment with water (Ctrl), 100 µM norspermidine (Nspd), or 100 µM spermidine (Spd).

Based on the results in Figures 3 and 4, we conclude that the MbaA phosphodiesterase and diguanylate cyclase domains are both required for MbaA to respond properly to polyamines to regulate V. cholerae biofilm dispersal. We propose a model in which elevated periplasmic norspermidine levels drive NspS to bind to MbaA. Consequently, MbaA phosphodiesterase activity is suppressed and the diguanylate cyclase activity dominates, which leads to c-di-GMP accumulation and commitment to the biofilm lifestyle (Figure 4F). In contrast, when spermidine is detected, or when periplasmic polyamine concentrations are low, NspS dissociates from MbaA. Consequently, MbaA diguanylate cyclase activity is suppressed and its phosphodiesterase activity dominates, which leads to a reduction in cytoplasmic c-di-GMP and favors biofilm dispersal (Figure 4F).

Polyamines control the V. cholerae biofilm lifecycle via periplasmic detection, not via import into the cytoplasm

Here, we investigate the role of PotD1 in controlling biofilm dispersal. Addition of norspermidine did not alter the non-dispersing phenotype of the ΔpotD1 mutant, whereas spermidine treatment drove premature biofilm dispersal and a reduction in peak biofilm biomass (Figure 5A, Video 1). Consistent with these findings, addition of norspermidine to the ΔpotD1 strain did not alter the c-di-GMP reporter output, whereas addition of spermidine reduced output signal, but only at the highest concentrations tested (Figure 5B). These biofilm lifecycle and reporter results show that PotD1-mediated import is not required for spermidine to modulate the biofilm lifecycle. Thus, we considered an alternative mechanism to underlie the V. cholerae ΔpotD1 phenotype. We hypothesized that endogenously produced norspermidine is secreted into the periplasm. In the case of WT V. cholerae, the equilibrium between norspermidine release and PotD1-mediated reuptake places MbaA into a partially liganded state, and as a consequence, MbaA exhibits modest net phosphodiesterase activity (Figure 5C). In the V. cholerae ΔpotD1 mutant that cannot reimport norspermidine, we predict that periplasmic norspermidine levels become elevated, NspS detects norspermidine, binds to MbaA, and promotes the MbaA diguanylate cyclase active state (Figure 5D). Hence, c-di-GMP is synthesized and biofilm dispersal is prevented. This hypothesis is consistent with our result showing that addition of norspermidine to the ΔpotD1 mutant does not cause any change in biofilm dispersal. Presumably, in the ΔpotD1 mutant, the NspS protein is already saturated due to elevated periplasmic levels of endogenously produced norspermidine. If this hypothesis is correct, norspermidine biosynthesis would be a requirement for biofilm formation because in the absence of norspermidine production, NspS would remain unliganded and MbaA would function as a constitutive phosphodiesterase (Figure 5E). Indeed, and consistent with previous results, the ΔnspC mutant that lacks the carboxynorspermidine decarboxylase enzyme NspC that is required for norspermidine synthesis failed to form biofilms (Figure 5F; Lee et al., 2009; McGinnis et al., 2009). Furthermore, the ΔnspC ΔpotD1 double mutant also failed to form biofilms, showing that endogenous norspermidine biosynthesis is required for the ΔpotD1 mutation to exert its effect (Figure 5F). Consistent with these interpretations, the ΔmbaA mutation is epistatic to the ΔpotD1 mutation as the ΔmbaA ΔpotD1 double mutant exhibited a dispersal phenotype indistinguishable from the single ΔmbaA mutant (Figure 5G). Administration of exogenous norspermidine or spermidine to the ΔmbaA ΔpotD1 double mutant (Figure 5H, Video 1) mimicked what occurred following addition to the single ΔmbaA mutant (Figure 3D): that is, essentially no response. Thus, information from externally supplied polyamines is transduced internally by MbaA and not via PotD1-mediated import. Lastly, to confirm that the observed ΔpotD1 phenotype was due to a defect in norspermidine transport, rather than a defect in periplasmic binding and sequestration of norspermidine, we deleted potA, encoding the ATPase that supplies the energy required for polyamine import through the PotABCD1 transporter (Kashiwagi et al., 2002). In the absence of PotA, PotD1 remains capable of binding polyamines, but no transport occurs. The ΔpotA mutant exhibited the identical dispersal defect as the ΔpotD1 mutant, demonstrating that transport of norspermidine, not sequestration by PotD1, is the key activity required for WT V. cholerae biofilm dispersal (Figure 5—figure supplement 1).

Figure 5 with 2 supplements see all
Polyamine import is not required for MbaA regulation of V. cholerae biofilm dispersal but is required to reduce external norspermidine levels.

(A) Quantitation of biofilm biomass over time measured by time-lapse microscopy following addition of water (Ctrl), 100 µM norspermidine, or 100 µM spermidine to the ΔpotD1 mutant. (B) c-di-GMP reporter output at the indicated polyamine concentrations for the ΔpotD1 strain. Relative reporter signal (% difference) is displayed as a heatmap (teal and purple represent the lowest and highest reporter output, respectively). Values are expressed as the percentage difference relative to the untreated WT strain, allowing comparisons to be made across all heatmaps in all figures in this article. The same color bar applies to all c-di-GMP reporter heatmaps in this article and for each condition, N = 3 biological replicates. Numerical values and associated SDs are available in Supplementary file 1. (C) Schematic of NspS-MbaA periplasmic detection of polyamines and polyamine import by PotD1 in WT V. cholerae. OM: outer membrane; IM: inner membrane. (D) Schematic of NspS-MbaA periplasmic detection of norspermidine together with the accumulation of elevated extracellular norspermidine in the V. cholerae ΔpotD1 mutant. (E) Schematic of NspS-MbaA activity in the ΔnspC mutant that is incapable of norspermidine biosynthesis. (F) Biofilm biomass over time for WT, the ΔnspC mutant, the ΔpotD1 mutant, and the ΔnspC ΔpotD1 double mutant. (G) As in (F) for WT, the ΔmbaA mutant, the ΔpotD1 mutant, and the ΔmbaA ΔpotD1 double mutant. (H) As in (A) for the ΔmbaA ΔpotD1 double mutant. All biofilm biomass data are represented as means normalized to the peak biofilm biomass of the WT strain or Ctrl condition, and in all cases, N = 3 biological and N = 3 technical replicates, ± SD (shaded). a.u.: arbitrary unit. (I) Concentration of norspermidine in cell-free culture fluids for the WT, ΔpotD1, and ΔnspC strains grown to OD600 = 2.0, measured by mass spectrometry. (J) Intracellular norspermidine levels normalized to wet cell pellet mass for the strains in (I). The strains used in (I) and (J) also contained a ΔvpsL mutation to abolish biofilm formation. Thus, all strains existed in the same growth state, which enabled comparisons between strains and, moreover, eliminated any possibility of polyamine sequestration by the biofilm matrix. For (I) and (J), N = 3 biological replicates, error bars represent SDs, and unpaired t-tests were performed for statistical analysis. n.s.: not significant; ***p<0.001.

To investigate the supposition that the ΔpotD1 strain possesses higher levels of periplasmic norspermidine than WT V. cholerae, one needs to isolate and measure polyamines in the periplasmic compartment. Unfortunately, we were unable to reliably obtain periplasmic compartment material. We reasoned, however, that some periplasmic norspermidine would diffuse into the extracellular environment, where it could be isolated and quantified, providing us a means to evaluate differences in periplasmic norspermidine levels between the WT and the ΔpotD1 mutant. At high cell density, approximately 25-fold more norspermidine was present in cell-free culture fluids collected from the ΔpotD1 mutant (average 2.3 µM) than in those prepared from the WT (average 90 nM) (Figure 5I). Norspermidine was nearly undetectable in culture fluids from the ΔnspC mutant (Figure 5I). There was no difference in norspermidine levels in whole-cell extracts prepared from WT (average 0.6 µmol/g) and the ΔpotD1 mutant (average 0.5 µmol/g) (Figure 5J). Thus, the difference we measured in norspermidine concentrations in the two extracellular fractions cannot be due to variations in norspermidine biosynthesis between the two strains. As expected, both the cell-free fluid and the whole-cell extract from the ΔnspC mutant were devoid of norspermidine and spermidine (Figure 5J, Figure 5—figure supplement 2), and consistent with previous reports (Lee et al., 2009), only trace spermidine could be detected in the WT intra- and extracellular preparations (Figure 5—figure supplement 2). For reference, we also measured the intra- and extracellular levels of additional polyamines from the same samples (Figure 5—figure supplement 2). Thus, our results argue against a cytoplasmic role for norspermidine in the biofilm lifecycle. Rather, our results indicate that the non-dispersing phenotype of the ΔpotD1 strain is due to elevated accumulation of periplasmic norspermidine stemming from the failure of the mutant to reuptake self-made norspermidine. The consequence is that, compared to WT V. cholerae, in the ΔpotD1 mutant, MbaA is biased in favor of c-di-GMP production and commitment to the biofilm lifestyle (Figure 5D).

MbaA and polyamine levels remain constant throughout the V. cholerae biofilm lifecycle

Our results demonstrate that WT V. cholerae is poised to respond to exogenous polyamines through the NspS-MbaA signaling circuit, presumably allowing V. cholerae to adapt to the species composition of its environment. We wondered if, under the conditions of our biofilm assay, MbaA receptor abundance or endogenously produced norspermidine/spermidine levels change over the course of the biofilm lifecycle, either or both of which could influence the normal biofilm growth to dispersal transition. To test these possibilities, we measured MbaA-3xFLAG protein levels and the concentrations of intra- and extracellular polyamines during biofilm formation (T = 5 hr) and after dispersal (T = 10 hr) in WT V. cholerae. MbaA abundance and the intracellular concentrations of norspermidine and spermidine did not fluctuate between the 5 hr and 10 hr timepoints (Figure 6A, B). The concentration of extracellular norspermidine did increase between 5 hr and 10 hr, from <10 nM to ~75 nM (Figure 6B); however, this range is far below the NspS-MbaA detection threshold (Figure 3C). Extracellular spermidine was nearly undetectable at both timepoints (Figure 6B). Measurements of additional polyamines from the same samples are shown in Figure 6—figure supplement 1. We take these results to mean that, in the absence of exogenously supplied polyamines, MbaA activity is constant and, in our experiments, the enzyme is modestly biased toward phosphodiesterase function throughout the biofilm lifecycle (as indicated by the ΔmbaA strain phenotypes shown in Figure 2B, C).

Figure 6 with 1 supplement see all
MbaA and intracellular polyamine levels remain constant throughout the biofilm lifecycle.

(A) Top panel: western blot showing MbaA-3xFLAG levels at 5 hr and 10 hr post inoculation. Bottom panel: the RpoA loading control. Data are representative of three biological replicates. (B) Measurements of intracellular (left panel) and extracellular (right panel) norspermidine (Nspd) and spermidine (Spd) levels at 5 hr and 10 hr post inoculation. Intracellular levels were normalized to wet cell pellet mass. N = 3 biological replicates, error bars represent SDs, and unpaired t-tests were performed for statistical analysis. n.s.: not significant; ***p<0.001.

Discussion

In this study, we investigated the effects of the polyamines norspermidine and spermidine on V. cholerae biofilm dispersal. Norspermidine and spermidine, which differ in structure only by one methylene group, mediate starkly opposing effects on the V. cholerae biofilm lifecycle: norspermidine inhibits and spermidine promotes biofilm dispersal. Both function through the NspS-MbaA circuit. Thus, the polyamine binding protein NspS must harbor the exquisite capability to assess the presence or absence of a single chemical moiety as norspermidine binding drives NspS interaction with MbaA, whereas binding to spermidine prevents this interaction. Here, we speculate on the possible biological significance of our findings. We suspect that norspermidine and spermidine act as ‘self’ and ‘other’ cues, respectively. Specifically, norspermidine is a rare polyamine in the biosphere, produced only by select organisms, namely V. cholerae and closely related marine vibrios (Hamana, 1997; Michael, 2018; Yamamoto et al., 1991). The observation that laboratory-grown WT V. cholerae releases little norspermidine, at least in part due to PotD1-mediated reuptake (Figure 5C), suggests that this system does not behave like a canonical quorum-sensing pathway. However, it is possible that norspermidine is secreted by V. cholerae under some environmental conditions, or by other vibrios, and V. cholerae detects the released norspermidine via NspS-MbaA, and its dispersal from biofilms is prevented. Thus, when close relatives are nearby, as judged by detection of the presence of norspermidine, V. cholerae elects to remain in its current biofilm niche. More speculative is the notion that norspermidine functions as a cue for phage-, predator-, or toxin-induced cell lysis. Specifically, if V. cholerae can detect norspermidine released by neighboring lysed V. cholerae cells, in response to the perceived danger, V. cholerae would remain in the protective biofilm state. Conversely, detection of spermidine, a nearly ubiquitously produced polyamine (Michael, 2018), could alert V. cholerae to the presence of competing or unrelated organisms. In this case, V. cholerae would respond by dispersing from biofilms and fleeing that locale. In a seemingly parallel scenario, we previously discovered that autoinducer AI-2, a universally produced quorum-sensing signal, also drove V. cholerae biofilm repression and premature dispersal, while CAI-1, the V. cholerae ‘kin’ quorum-sensing autoinducer, did not (Bridges and Bassler, 2019). Together, our findings suggest that V. cholerae possesses two mechanisms to monitor its own numbers (norspermidine and CAI-1) and two mechanisms to take a census of unrelated organisms in the environment (spermidine and AI-2), and it responds by remaining in the biofilm state when closely related species are in the majority and by exiting the biofilm state when the density of non-related organisms is high, presumably to escape competition/danger.

We found that periplasmic polyamine detection by NspS-MbaA, but not polyamine import, controls the biofilm dispersal program and both the MbaA EVL and SGDEF domains are required for the V. cholerae response to polyamines. Despite previous work suggesting that MbaA functions exclusively as a phosphodiesterase and that its SGDEF domain, which possesses a serine substitution in the active site relative to the canonical GGDEF active site, is inert with respect to c-di-GMP synthesis, our data suggest that when norspermidine is present, MbaA does synthesize c-di-GMP (Figure 3C, Figure 4C), as has also been shown for other SGDEF proteins (Pérez-Mendoza et al., 2011). Therefore, MbaA is likely a bifunctional protein capable of both c-di-GMP synthesis and degradation. We wonder how such an arrangement benefits V. cholerae in its response to polyamines. We speculate that the ability of MbaA to inversely alter c-di-GMP levels in response to two discrete cues coordinates polyamine signaling and enables more rapid and greater magnitude changes in c-di-GMP levels than would be achievable if two separate receptors existed, each harboring only a single catalytic activity and each responsive to only one polyamine.

An analogous bifunctional arrangement exists for the quorum-sensing two-component receptor LuxN from Vibrio harveyi. LuxN is an inner membrane receptor that harbors a periplasmic sensory domain responsible for detection of a self-made small molecule cue, a homoserine lactone agonist with a four-carbon acyl tail (Bassler et al., 1994). The ligand encodes species-specific information about cell population density. Competing bacterial species that co-occupy the niche in which V. harveyi resides produce similar acyl homoserine lactone signal molecules, and notably, those molecules differ only in the acyl tail length or decoration (Papenfort and Bassler, 2016). The tails of the molecules produced by the competitors usually possess more than four carbons, and they act as potent LuxN antagonists. With respect to mechanism, the self-made agonist drives LuxN into phosphatase mode while the antagonists induce LuxN to act as a kinase (Ke et al., 2015). Thus, as in the MbaA circuit, binding of the different ligands to LuxN propels opposing enzymatic activities in the cytoplasm. We speculate that ligand-driven alternations between opposing enzymatic activities could be an underappreciated mechanism that sensors employ to transmit species-specific and species-non-specific information into discrete changes in downstream behaviors.

In addition to conveying information about the species composition of the vicinal community, the MbaA SGDEF and EVL domains could also participate in dimerization and/or allosteric binding to c-di-GMP or other metabolites to mediate feedback regulation of the opposing MbaA activities. Parallel examples exist, here we present one: Caulobacter crescentus contains a GGDEF/EAL protein called CC3396, in which the GGDEF domain is non-catalytic but it nonetheless binds to GTP, which allosterically activates the phosphodiesterase activity in the neighboring EAL domain (Christen et al., 2005). A feedback mechanism could provide MbaA with the ability to integrate the information encoded in extracellular polyamine blends with cytoplasmic cues, such as the metabolic state of the cell or the current cytoplasmic c-di-GMP level. Taken together, the double ligand detection capability linked to the dual catalytic activities of the NspS-MbaA circuit allows V. cholerae to distinguish between remarkably similar ligands and, in response, have the versatility to convey distinct information into the cell to alter the biofilm lifecycle.

In summary, four signaling pathways have now been defined that feed into the regulation of V. cholerae biofilm dispersal: starvation (RpoS) (Singh et al., 2017), quorum sensing (via CAI-1 and AI-2) (Bridges and Bassler, 2019; Singh et al., 2017), the recently identified DbfS/DbfR cascade (ligand unknown) (Bridges et al., 2020), and through the current work, polyamine signaling via NspS-MbaA. We propose that integrating the information contained in these different stimuli into the control of biofilm dispersal endows V. cholerae with the ability to successfully evaluate multiple features of its fluctuating environment prior to committing to the launch of this key lifestyle transition that, ultimately, impinges on its overall survival. Because V. cholerae biofilm formation and biofilm dispersal are intimately connected to cholera disease and its transmission, we suggest that deliberately controlling the biofilm lifecycle, possibly via synthetic strategies that target polyamine signal transduction via NspS-MbaA, could be a viable therapeutic strategy to ameliorate disease.

Materials and methods

Bacterial strains, reagents, and imaging assays

Request a detailed protocol

The V. cholerae strain used in this study was WT O1 El Tor biotype C6706str2. Antibiotics were used at the following concentrations: polymyxin B, 50 μg/mL; kanamycin, 50 μg/mL; spectinomycin, 200 μg/mL; chloramphenicol, 1 μg/mL; and gentamicin, 15 μg/mL. Strains were propagated for cloning purposes in lysogeny broth (LB) supplemented with 1.5% agar or in liquid LB with shaking at 30°C. For biofilm dispersal analyses, lux measurements, c-di-GMP reporter quantitation, and norspermidine measurements, V. cholerae strains were grown in M9 medium supplemented with 0.5% dextrose and 0.5% casamino acids. All strains used in this work are reported in the Key resources table. Compounds were added from the onset of biofilm initiation. Norspermidine (Millipore Sigma, I1006-100G-A) and spermidine (Millipore Sigma, S2626-1G) were added at the final concentrations designated in the figures. The biofilm lifecycle was measured using time-lapse microscopy as described previously (Bridges and Bassler, 2019). All plots were generated using ggplot2 in R. Light production driven by the vpsL promoter was monitored as described previously (Bridges et al., 2020). Results from replicates were averaged and plotted using ggplot2 in R.

DNA manipulation and strain construction

Request a detailed protocol

All strains generated in this work were constructed by replacing genomic DNA with DNA introduced by natural transformation as previously described (Bridges et al., 2020). PCR and Sanger sequencing were used to verify correct integration events. Genomic DNA from recombinant strains was used for future co-transformations and as templates for PCR to generate DNA fragments, when necessary. See Key resources table for primers and g-blocks (IDT) used in this study. Gene deletions were constructed in frame and eliminated the entire coding sequences. The exceptions were mbaA and nspS, which overlap with adjacent genes, so an internal portion of each gene was deleted, ensuring that adjacent genes were not perturbed. To construct mbaA-3xFLAG at the native mbaA locus, gene synthesis was used to preserve the coding sequence of the downstream gene. To achieve this, the overlapping region was duplicated. In the duplication, the start codon of the downstream gene was disabled by mutation. The coding sequencing of mbaA was preserved. The DNA specifying 3xFLAG was introduced immediately upstream of the mbaA stop codon. All strains constructed in this study were verified using the Genewiz sequencing service.

Western blotting

Request a detailed protocol

Cultures of strains carrying MbaA-3xFLAG and relevant catalytic site variants were collected at OD600 = 1.0 and subjected to centrifugation for 1 min at 13,000 rpm. The pellets were flash frozen, thawed for 5 min at 25oC, and subsequently chemically lysed by resuspending to OD600 = 1.0 in 75 μL Bug Buster (Novagen, #70584-4) supplemented with 0.5% Triton-X, 50 μg/mL lysozyme, 25 U/mL benzonase nuclease, and 1 mM phenylmethylsulfonyl fluoride for 10 min at 25oC. Lysates were solubilized in 1× SDS-PAGE buffer for 1 hr at 37°C. Samples were loaded into 4–20% Mini-Protein TGX gels (Bio-Rad). Electrophoresis was carried out at 200 V. Proteins were transferred from the gels to PVDF membranes (Bio-Rad) for 50 min at 4oC at 100 V in 25 mM Tris base, 190 mM glycine, 20% methanol. Following transfer, membranes were blocked in 5% milk in PBST (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, and 0.1% Tween) for 1 hr, followed by three washes with PBST. Subsequently, membranes were incubated for 1 hr with a monoclonal Anti-FLAG-Peroxidase antibody (Millipore Sigma, #A8592) at a 1:5000 dilution in PBST with 5% milk. After washing six times with PBST for 5 min each, membranes were exposed using the Amersham ECL western blotting detection reagent (GE Healthcare). For the RpoA loading control, the same protocol was followed except that the primary antibody was Anti-Escherichia coli RNA Polymerase α (Biolegend, #663104) used at a 1:10,000 dilution and the secondary antibody was an Anti-Mouse IgG HRP conjugated antibody (Promega, #W4021) used at a 1:10,000 dilution.

Extraction and measurements of polyamines

Request a detailed protocol

To measure intracellular polyamines, previously established techniques were followed with slight modifications (McGinnis et al., 2009). For planktonic cell measurements, V. cholerae strains were grown in 5 mL of M9 medium containing glucose and casamino acids with constant shaking at 30oC. 2.0 OD600 equivalents of V. cholerae cells were collected by centrifugation for 1 min at 13,000 rpm from cultures that had been grown to OD600 ~2.0. Cells were washed once with 1× PBS and subsequently weighed and resuspended in 100 µL PBS. The resuspended cells were lysed by 10 freeze-thaw cycles in liquid nitrogen. After the final freeze-thaw cycle, samples were subjected to bath sonication for 20 s (Fisher Scientific, FS30) and subsequently diluted to 100 mg/mL in 1× PBS. To precipitate proteins, 100 µL of 50% trichloroacetic acid was added to 500 µL of lysate and the mixtures were incubated for 5 min. Precipitated material was pelleted by centrifugation for 5 min at 13,000 rpm. 500 µL of the resulting clarified supernatants were subjected to benzoylation as described below. For measurements of polyamines in cell-free culture fluids, the culture fluids from the samples described above were collected following the centrifugation step. Remaining cells were removed by filtration with 0.45 µm filters (Millex-HV), proteins were precipitated as above, and the benzoylation derivatization was performed. For measurements of polyamine concentrations in static cultures at the 5 hr and 10 hr timepoints, the procedure was the same as above with the following modifications: 10 mL cultures were incubated at 30°C without shaking to achieve conditions favoring the biofilm lifecycle. In the case of the 5 hr cultures, samples were briefly mixed by vortex with sterile 1 mm glass beads to dislodge biofilms prior to centrifugation. Cell pellets from both the 5 hr and 10 hr samples were resuspended in 1× PBS at 40 mg/mL, proteins were precipitated, and 500 µL was used for derivatization.

Samples were derivatized with benzoyl chloride as previously described, except that the amount of benzoyl chloride was doubled to ~80 µmol, and benzylated polyamines were resuspended in 200 µL of the mobile phase (Morgan, 1998). In all cases, medium blanks and polyamine standards were simultaneously derivatized and analyzed to generate calibration curves. HPLC was performed on a Shimadzu UFLC system with PAL autoinjector. 30 min gradient chromatography separation was performed using solvent A (10% methanol/90% water) and solvent B (95% methanol/5% water) on an ACE Ultracore 2.5 SuperC18 1.0 × 50 mm column with 63 µL/min flow rate at a column temperature of 54°C. Mass spectrometry was performed using an Orbitrap XL mass spectrometer (Thermo) with an APCI ionization source in positive mode. The parent ion (MS1) was detected in the Orbitrap with 100,000 mass resolution, and fragment ions (MS2) were detected in the ion trap. Parameters were as follows: the vaporizer temperature was 270°C, the sheath gas flow rate was 18 a.u., the auxiliary gas flow rate was 5 a.u., the sweep gas flow rate was 5 a.u., the discharge current was 9 mA, the capillary temperature was 250°C, the capillary voltage was 36 V, and the tube lens was 72 V. Skyline software (University of Washington) was used to analyze results.

c-di-GMP reporter assays

Request a detailed protocol

The c-di-GMP reporter has been described (Zamorano-Sánchez et al., 2019; Zhou et al., 2016). To measure relative reporter output for each condition, 100 µL of V. cholerae cultures were back diluted to OD600 = 0.0002 following overnight growth. Cultures were dispensed into 96-well plates containing the indicated polyamines, and the plates were covered in breathe-easy membranes to prevent evaporation. Samples were incubated overnight at 30°C with shaking. The following morning, the breathe-easy membranes were removed and fluorescence measurements were obtained using a BioTek Synergy Neo2 Multi-Mode reader. For AmCyan, the excitation wavelength was 440 ± 20 nm and emission was detected at 490 ± 20 nm; and for TurboRFP, the excitation wavelength was 530 ± 20 nm and the emission was 575 ± 20 nm. The c-di-GMP-regulated TurboRFP fluorescence was divided by the constitutive AmCyan fluorescence to yield the relative fluorescence intensity (RFI). To facilitate comparisons between strains and conditions, RFIs were subsequently normalized to the untreated V. cholerae WT RFI and the data are expressed as the percentage differences (denoted relative reporter signal). All results were obtained in biological triplicate, and data analysis and plotting were performed in R.

Data availability

All data generated and analyzed in this study are included in the manuscript and supporting files. Source data files have been provided in Zenodo (https://doi.org/10.5281/zenodo.4651348).

The following data sets were generated
    1. Bridges AA
    2. Bassler BL
    (2021) Zenodo
    Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals.
    https://doi.org/10.5281/zenodo.4651348

References

  1. Book
    1. Morgan DML
    (1998)
    Determination of polyamines as their benzoylated derivatives by HPLC
    In: Morgan D. M. L, editors. Polyamine Protocols, Methods in Molecular Biology. Humana Press. pp. 111–118.

Decision letter

  1. Michael T Laub
    Reviewing Editor; Massachusetts Institute of Technology, United States
  2. Gisela Storz
    Senior Editor; National Institute of Child Health and Human Development, United States
  3. Anthony Michael
    Reviewer
  4. Mark Mandel
    Reviewer

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This paper presents evidence that the polyamines spermidine and norspermidine can inversely impact the dispersal of Vibrio cholerae biofilms through the protein MbaA, which contains a periplasmic domain that may interface with a possibly direct polyamine sensor, NspS. The paper presents compelling data to support the notion that the cyclic-di-GMP synthetase and phosphodiesterase domains of MbaA are ultimately responsible for transducing polyamine signals in the periplasm into changes in biofilm formation. The work indicates that external polyamines may function to provide self/non-self information, similar to quorum sensing systems.

Decision letter after peer review:

Thank you for submitting your article "Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Anthony Michael (Reviewer #2); Mark Mandel (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

This paper presents evidence that the polyamines spermidine and norspermidine can inversely impact the dispersal of Vibrio cholerae biofilms through the protein MbaA, which contains a periplasmic domain that may interface with a possibly direct polyamine sensor, NspS. The paper is easy to follow and presents some compelling data to support the notion that the cyclic-di-GMP synthetase and phosphodiesterase domains of MbaA are ultimately responsible for transducing polyamine signals in the periplasm into changes in biofilm formation. The work indicates that external polyamines may function to provide self/non-self information, similar to quorum sensing systems. There were several major concerns raised, as summarized below. In particular, there was concern from all three reviewers about how much polyamine-based signaling contributes to the formation of biofilms by wild-type cells, especially the self-produced norspermidine. In short, there's currently confusion about whether polyamines only impact biofilms when added exogenously or whether the endogenously produced/secreted levels can and do impact biofilms. Measuring these polyamines in biofilm cultures will be essential to a revision.

Essential revisions:

Experimental issues:

The deletion mutants (e.g. in Figure 2) need to be complemented.

The evidence for the periplasmic sensing model in the ΔpotD1 mutant is strong, but could be further strengthened by an intervention that blocks the proposed norspermidine export. If there is a way to interrupt endogenous norspermidine synthesis and/or export to the periplasm, this would add further support for the model in Figure 5CD to explain the role of PotD1. Similarly, a PotD1 allele that is still present in the periplasm but is nonfunctional for import would provide evidence that the key issue is flux/import, and not sequestration for PotD1.

The fact that the mbaA(SGDEF)* mutant still accumulates biomass at the same rate up to 8 hr as the wild-type with the same max value suggests that this pathway doesn't become relevant until later in biofilm formation. But, spermidine at high levels can inhibit early on (Figure 3A). To me, these two findings aren't reconciled in the authors' model (Figure 4F) which would lead one to believe that CDG synthesis by MbaA is critical for biofilm formation, but it's not (see Figure 4A) – to be clear, I recognize that the paper's title and abstract focus on dispersal, but the model leads one to think that the pathway being studied here is important throughout the biofilm development process. Maybe more generally, the issue is about how spermidine and norspermidine impact the normal development of a wild-type biofilm, if at all. It's not revealed until the Discussion that norspermidine isn't likely secreted at appreciable levels (something that should definitely be noted in the Introduction), so while exogenous addition can impact biofilms, the lack of secreted norspermidine makes it sound like it's actually not so critical to our understanding of WT biofilms and the behavior of WT biofilms documented in Figure 2. Following on this point above, I have two questions that seem critical to assessing how polyamines impact biofilm development: (i) What's the level of spermidine produced and secreted? If the dispersal is normally driven by spermidine, its levels should accumulate extracellularly or periplasmically at the time biofilms being to disperse. (ii) What's the biofilm phenotype of a mutant that cannot synthesize spermidine or norspermidine or both? It could be that mutants that can't synthesize polyamines behave like the WT and that neither is detectable in the periplasm/secreted fraction – such results wouldn't negate or diminish the conclusion that these polyamines can impact biofilm formation when added exogenously (which the Discussion speculates on), but it would mean that the usual pattern of biofilm mass accumulation and then drop off noted in Figure 2B isn't driven by the polyamines. At the very least, the initial accumulation seems very unlikely to be driven by norspermidine if it can't be detected.

Following on the point above, the group of Karatan, using HPLC, has repeatedly failed to detect extracellular norspermidine in the spent growth medium of V. cholerae planktonic and biofilm cultures (refs. Wotanis et al., 2017 and uncited Parker et al., (2012) FEMS Microbiol Lett 329, 19-27). The current authors have used LC-MS/MS to determine that spent growth medium from planktonic potD1 gene deletion cultures contain 15 times more norspermidine than the parental wildtype strain. The authors need to provide a quantitative analysis of the absolute levels of norspermidine produced by biofilm cultures and assess whether it exceeds the minimum amount required to see effects when exogenously adding norspermidine.

The hypothesis of the authors – that self-produced norspermidine in the Δ-potD1 mutant accumulates in the periplasm and elicits biofilm effects due to its periplasmic concentration, is elegant but requires two suppositions: firstly, norspermidine is secreted by the cell into the periplasm, and that the periplasmic concentration of norspermidine must be above a threshold that results in elicitation of biofilm and dispersal effects. Norspermidine is presumably able to freely diffuse out of the periplasm into the external medium via porins. Given the time scales involved, the concentration of norspermidine would be likely to equilibrate either side of the outer membrane. Therefore, either the concentration of norspermidine in the external cell-free medium must be above a threshold level that elicits biofilm effects, as discussed above. Or, norspermidine must be sequestered in the periplasm. How might sequestration of the norspermidine in the periplasm occur? The Karatan group showed that the cell-free spent medium from V. cholerae biofilm cultures contained 2 mM cadaverine, whereas the sterile cell-free growth medium contained only 3 μM cadaverine. This indicates that the biofilm cells were undergoing acid stress, which induces the cadaverine-producing and exporting cadAB system (lysine decarboxylase and lysine/cadaverine antiporter) but more relevantly, endogenously-produced cadaverine has been shown to block outer membrane porin channels, even if produced under pH neutral conditions (Samartzidou and Delcour (1999) "Excretion of endogenous cadaverine leads to a decrease in porin-mediated outer membrane permeability", J. Bacteriol., 181, 791-798). Production and excretion of cadaverine by CadAB during biofilm formation could prevent self-produced norspermidine diffusing out of the periplasm. In the WT cells, any periplasmic norspermidine could be reacquired by the cell through the potABCD transporter, whereas in the Δ-potD1 mutant, a cadaverine block of porins might result in accumulation of periplasmic norspermidine. Questions to the authors: (1) What was the concentration of cadaverine in the WT and Δ-potD1 cell cultures and cell-free spent medium? (2) What was the concentration of putrescine, spermidine and N-acetylnorspermidine? Presumably this data is part of the original LC-MS/MS output. (3) The authors' polyamine data was obtained with planktonic cultures containing an additional deletion of the vps1 gene that eliminates the biofilm matrix. Did the authors measure norspermidine levels in cell-free spent medium from biofilm cultures? If not, it would be very relevant to the authors' hypothesis to measure norspermidine and cadaverine in the cell-free spent medium of WT and Δ-potD1 biofilm cultures (that are +vps1) and compare with sterile, cell-free medium. The blockage of porin permeability in the Samartzidou and Delcours study occurred when external cadaverine concentration had reached only 0.2 mM, whereas the Karatan group found 2 mM cadaverine in the external medium, suggesting that in the V. cholerae biofilm cells, cadaverine-blocked porins would prevent free diffusion of norspermidine out of the periplasm. Thus, cadaverine measurements would be very relevant to the authors' conclusions.

Scholarship issues:

A more thorough scholarly analysis of what is known about NspS (versus proposed here) would bolster the role for NspS in the authors' model. The current manuscript does not describe the key literature on NspS. In particular, the sensing of polyamines by MbaA through NspS-polyamine binding and direct binding of NspS to MbaA seems well-supported by literature that could be described.

The paper generally lacks context and does not discuss in adequate detail the general biological roles for polyamines (including cytoplasmically), what's already known about their roles in biofilm formation (in vibrios or other organisms like B. subtilis), or how they are synthesized. Without this sort of context, I think the paper will be difficult to access or appreciate for the non-expert/non-biofilm researcher.

It is surprising that the authors did not discuss and cite the Sobe et al., paper, "Spermine inhibits Vibrio cholerae biofilm formation through the NspS-Mba polyamine signaling system" (2017) J. Biol. Chem. 292, 17025-17036. This paper discusses the concept of self/non-self mediated by polyamines and makes a persuasive argument that spermine (a primarily eukaryotic polyamine) is a more likely signal than spermidine, especially for a human pathogen.

The authors did not measure cyclic-di-GMP directly and did not measure polyamine transport directly. It would therefore be prudent to make less categorically emphatic statements about eg., diguanylate cyclase activity line 339 "our data show that MbaA does synthesize c-di-GMP when norspermidine is present (Figure 3C, Figure 4C). In fact, c-di-GMP was not measured and the diguanylate cyclase activity of MbaA was not biochemically assayed. Similarly, for norspermidine uptake – in the abstract ("Biofilm dispersal fails in the absence of PotD1 because reuptake of endogenously produced norspermidine does not occur..") and line 109. At this stage, more tentative language would be appropriate.

Regarding self/non-self, I had difficulty integrating the discussion proposing that "self" molecules promote biofilm with a role for biofilm dispersal in cholera transmission (when, presumably, the "self" molecules would be at high abundance). I think a more sophisticated discussion of the issue here would be helpful, given that this is the lab that has defined the relevant QS signaling, has distinguished surface biofilms vs. liquid aggregates, etc.

https://doi.org/10.7554/eLife.65487.sa1

Author response

Essential revisions:

Experimental issues:

The deletion mutants (e.g. in Figure 2) need to be complemented.

We thank the reviewers for pointing out this issue. As requested, we expressed mbaA and potD1 from an ectopic locus on the V. cholerae chromosome. Introduction of each gene in the corresponding deletion mutant restored the WT biofilm lifecycle behavior. These results are provided in new Figure 2—figure supplement 1.

The evidence for the periplasmic sensing model in the ΔpotD1 mutant is strong, but could be further strengthened by an intervention that blocks the proposed norspermidine export. If there is a way to interrupt endogenous norspermidine synthesis and/or export to the periplasm, this would add further support for the model in Figure 5CD to explain the role of PotD1. Similarly, a PotD1 allele that is still present in the periplasm but is nonfunctional for import would provide evidence that the key issue is flux/import, and not sequestration for PotD1.

We agree that the suggested experiments would confirm our model. Unfortunately, we do not know the identity of the exporter that transports norspermidine from the cytoplasm to the periplasm, so we cannot perform that test. However, we can disrupt norspermidine biosynthesis via deletion of nspC, which has the added benefit of eliminating norspermidine export to the periplasm. Consistent with our model (and as has been shown previously by other groups), the ΔnspC strain failed to form biofilms (shown in new Figure 5F; discussed in text lines 319-327). Furthermore, we made the ΔnspC ΔpotD1 double mutant and it also failed to form biofilms, demonstrating that norspermidine production is epistatic to norspermidine import (shown in new Figure 5F).

To distinguish between polyamine import and PotD1-mediated sequestration of norspermidine, we deleted potA. PotABCD is the transport apparatus responsible for spermidine/norspermidine import. The role of the PotA ATPase is to supply the energy required for transport. Thus, in the absence of PotA, PotD1 remains capable of binding polyamines, yet transport does not occur. The ΔpotA mutant exhibited the identical dispersal defect as the ΔpotD1 mutant validating that transport of norspermidine, not sequestration by PotD1, is the key activity required for biofilm dispersal. The results are reported in new Figure 5—figure supplement 1, along with text in lines 333-340.

The fact that the mbaA(SGDEF)* mutant still accumulates biomass at the same rate up to 8 hr as the wild-type with the same max value suggests that this pathway doesn't become relevant until later in biofilm formation. But, spermidine at high levels can inhibit early on (Figure 3A). To me, these two findings aren't reconciled in the authors' model (Figure 4F) which would lead one to believe that CDG synthesis by MbaA is critical for biofilm formation, but it's not (see Figure 4A) – to be clear, I recognize that the paper's title and abstract focus on dispersal, but the model leads one to think that the pathway being studied here is important throughout the biofilm development process. Maybe more generally, the issue is about how spermidine and norspermidine impact the normal development of a wild-type biofilm, if at all.

We thank the referees for this set of comments/suggestions. Here, we respond to them one by one for clarity:

We take the referee’s first question to be: what level of control does the MbaA signaling pathway exert over the biofilm lifecycle in WT V. cholerae in the absence of exogenously supplied polyamines? As the reviewer notes, in the case of the WT strain under conditions in which exogenous polyamines are not supplied, it appears, based on our results, that MbaA plays a minor role in driving progression of the biofilm lifecycle because the ∆mbaA mutant forms nearly WT biofilms and only exhibits a modest dispersal defect (Figure 2B). However, we have now analyzed and included results for the ΔnspC strain (discussed above) which is unable to synthesize and secrete norspermidine (Figure 5F). Due to the absence of endogenously-produced norspermidine, the ΔnspC strain does not form biofilms, as has also been demonstrated by other groups. The ΔnspC mutant phenotype shows that, in WT, when MbaA is present, periplasmic norspermidine is required to suppress the MbaA phosphodiesterase activity, which in turn, allows biofilms to form. Thus, in WT, the MbaA pathway has a major role as it is required for the normal biofilm lifecycle to take place.

It's not revealed until the Discussion that norspermidine isn't likely secreted at appreciable levels (something that should definitely be noted in the Introduction), so while exogenous addition can impact biofilms, the lack of secreted norspermidine makes it sound like it's actually not so critical to our understanding of WT biofilms and the behavior of WT biofilms documented in Figure 2.

As requested, we have clarified this point. We have included the following text in the Introduction (lines 92-96): “V. cholerae produces intracellular norspermidine, however, norspermidine has not been detected in cell-free culture fluids of laboratory grown strains (Parker et al., 2012). Thus, if norspermidine does indeed enable V. cholerae to take a census of ‘self’, it is apparently not via a canonical quorum-sensing type mechanism.”

In the discussion, we now write (lines 395-400), “The observation that laboratory grown WT V. cholerae releases little norspermidine, at least in part due to PotD1-mediated reuptake (Figure 5C), suggests that this system does not behave like a canonical quorum sensing pathway. However, it is possible that norspermidine is secreted by V. cholerae under some environmental conditions, or by other vibrios, and V. cholerae detects the released norspermidine via NspS-MbaA, and its dispersal from biofilms is prevented.”

Following on this point above, I have two questions that seem critical to assessing how polyamines impact biofilm development: (i) What's the level of spermidine produced and secreted? If the dispersal is normally driven by spermidine, its levels should accumulate extracellularly or periplasmically at the time biofilms being to disperse.

We appreciate this insight and suggestion. We performed the requested analyses. Please see lines 367-383 and the new Figure 6B. In brief, in the revised manuscript, we now provide quantitation of intracellular and extracellular spermidine and norspermidine concentrations at two timepoints – one prior to (5 h) and one after (10 h) biofilm dispersal. We find that, consistent with previous reports, V. cholerae makes almost no spermidine under all conditions tested. Furthermore, no substantial fluctuations in norspermidine levels (intracellular or extracellular) occurred between the timepoints that could be responsible for driving biofilm dispersal. We also considered the possibility that MbaA levels could change over the biofilm lifecycle, which could alter MbaA output activity. We therefore quantified MbaA-3xFLAG levels prior to (5 h) and after (10 h) biofilm dispersal (new Figure 6A). We found that MbaA levels remained constant. Our conclusion is that MbaA is poised to regulate the biofilm lifecycle in response to exogenous polyamines. Moreover, in the WT strain in the absence of environmentally-supplied polyamines, MbaA activity is constant and biased toward phosphodiesterase function throughout the biofilm lifecycle.

(ii) What's the biofilm phenotype of a mutant that cannot synthesize spermidine or norspermidine or both? It could be that mutants that can't synthesize polyamines behave like the WT and that neither is detectable in the periplasm/secreted fraction – such results wouldn't negate or diminish the conclusion that these polyamines can impact biofilm formation when added exogenously (which the Discussion speculates on), but it would mean that the usual pattern of biofilm mass accumulation and then drop off noted in Figure 2B isn't driven by the polyamines. At the very least, the initial accumulation seems very unlikely to be driven by norspermidine if it can't be detected.

We thank the referee for this suggestion. We have now assayed the biofilm lifecycle of the ∆nspC mutant that cannot synthesize norspermidine (new Figure 5F; discussed in lines 319-327). No biofilm formation occurs in this mutant demonstrating that, for normal biofilm development to occur, periplasmic norspermidine is required to suppress the MbaA phosphodiesterase activity (as discussed above in response to point 3). As noted above, we now also included a Results section demonstrating that changes in MbaA activity do not drive biofilm dispersal in the absence of exogenously supplied polyamines (new Figure 6; discussed in lines 367-383).

Following on the point above, the group of Karatan, using HPLC, has repeatedly failed to detect extracellular norspermidine in the spent growth medium of V. cholerae planktonic and biofilm cultures (refs. Wotanis et al., 2017 and uncited Parker et al., (2012) FEMS Microbiol Lett 329, 19-27). The current authors have used LC-MS/MS to determine that spent growth medium from planktonic potD1 gene deletion cultures contain 15 times more norspermidine than the parental wildtype strain. The authors need to provide a quantitative analysis of the absolute levels of norspermidine produced by biofilm cultures and assess whether it exceeds the minimum amount required to see effects when exogenously adding norspermidine.

We thank the reviewer for this suggestion. We have now repeated our measurements and we provide molar concentrations for all of the polyamines quantified by LC-MS/MS (see new Figure 5I, J and text lines 341-364). We write, “At high cell density, approximately 25-fold more norspermidine was present in cell-free culture fluids collected from the ΔpotD1 mutant (average 2.3 µM) than in those prepared from the WT (average 90 nM) (Figure 5I). […] There was no difference in norspermidine levels in whole cell extracts prepared from WT (average 0.6 µmol/g) and the ΔpotD1 mutant (average 0.5 µmol/g) (Figure 5J).”

Regarding our measurements from WT biofilm cultures, we now write, lines 376-379 “The concentration of extracellular norspermidine did increase between 5 h and 10 h, from <10 nM to ~75 nM (Figure 6B), however this range is far below the NspS-MbaA detection threshold (Figure 3C). Extracellular spermidine was nearly undetectable at both timepoints (Figure 6B).”

The hypothesis of the authors – that self-produced norspermidine in the Δ-potD1 mutant accumulates in the periplasm and elicits biofilm effects due to its periplasmic concentration, is elegant but requires two suppositions: firstly, norspermidine is secreted by the cell into the periplasm, and that the periplasmic concentration of norspermidine must be above a threshold that results in elicitation of biofilm and dispersal effects. Norspermidine is presumably able to freely diffuse out of the periplasm into the external medium via porins. Given the time scales involved, the concentration of norspermidine would be likely to equilibrate either side of the outer membrane. Therefore, either the concentration of norspermidine in the external cell-free medium must be above a threshold level that elicits biofilm effects, as discussed above. Or, norspermidine must be sequestered in the periplasm. How might sequestration of the norspermidine in the periplasm occur? The Karatan group showed that the cell-free spent medium from V. cholerae biofilm cultures contained 2 mM cadaverine, whereas the sterile cell-free growth medium contained only 3 μM cadaverine. This indicates that the biofilm cells were undergoing acid stress, which induces the cadaverine-producing and exporting cadAB system (lysine decarboxylase and lysine/cadaverine antiporter) but more relevantly, endogenously-produced cadaverine has been shown to block outer membrane porin channels, even if produced under pH neutral conditions (Samartzidou and Delcour (1999) "Excretion of endogenous cadaverine leads to a decrease in porin-mediated outer membrane permeability", J. Bacteriol., 181, 791-798). Production and excretion of cadaverine by CadAB during biofilm formation could prevent self-produced norspermidine diffusing out of the periplasm. In the WT cells, any periplasmic norspermidine could be reacquired by the cell through the potABCD transporter, whereas in the Δ-potD1 mutant, a cadaverine block of porins might result in accumulation of periplasmic norspermidine. Questions to the authors: (1) What was the concentration of cadaverine in the WT and Δ-potD1 cell cultures and cell-free spent medium? (2) What was the concentration of putrescine, spermidine and N-acetylnorspermidine? Presumably this data is part of the original LC-MS/MS output. (3) The authors' polyamine data was obtained with planktonic cultures containing an additional deletion of the vps1 gene that eliminates the biofilm matrix. Did the authors measure norspermidine levels in cell-free spent medium from biofilm cultures? If not, it would be very relevant to the authors' hypothesis to measure norspermidine and cadaverine in the cell-free spent medium of WT and Δ-potD1 biofilm cultures (that are +vps1) and compare with sterile, cell-free medium. The blockage of porin permeability in the Samartzidou and Delcours study occurred when external cadaverine concentration had reached only 0.2 mM, whereas the Karatan group found 2 mM cadaverine in the external medium, suggesting that in the V. cholerae biofilm cells, cadaverine-blocked porins would prevent free diffusion of norspermidine out of the periplasm. Thus, cadaverine measurements would be very relevant to the authors' conclusions.

We thank the referee for these insightful comments. To test the referee’s hypothesis that cadaverine blockage of porins causes norspermidine trapping in the periplasm, we performed several experiments. Our data are provided in Author response image 1. We find no evidence for cadaverine export in controlling extracellular norspermidine levels or in driving the biofilm lifecycle.

In the revised manuscript, we now provide quantitation of cadaverine, putrescine, spermidine, and norspermidine levels in all of our polyamine measurements. Those data are provided in new Figure 5 I, J; new Figure 5—figure supplement 2; new Figure 6B; and new Figure 6—figure supplement 1.

Regarding cadaverine: First, in WT planktonic, shaken cultures at high cell density, cadaverine was present at 0.5 µM in cell-free culture fluids (new Figure 5—figure supplement 2B; middle panel). In biofilm cells grown under static conditions, concentrations of both intracellular and extracellular cadaverine were low at the 5 h timepoint and dramatically increased at 10 h (new Figure 6—figure supplement 1). These results are consistent with those in the Karatan manuscript that demonstrated higher cadaverine levels for statically grown biofilm cells than for cells grown in shaken culture. Both sets of results suggest that, at late times, acid stress occurs in the static cultures, cadAB is induced, and cadaverine accumulates. Our temporal results, however, are inconsistent with the referee’s hypothesis that cadaverine blockage of porin channels leads to elevated norspermidine and subsequent changes in NspS-MbaA signaling. Specifically, cadaverine is in low abundance at the 5 h timepoint, when biofilm formation is favored. Cadaverine concentration increases by 10 h, after dispersal has occurred. This timing suggests that, if cadaverine had blocked porins, norspermidine would have accumulated in the periplasm by the 10 h timepoint, promoting biofilm formation, however, that does not occur. Rather, we observe biofilm dispersal.

We further examined the relationship between cadaverine and the biofilm lifecycle by constructing a ΔcadB mutant that lacks the lysine/cadaverine antiporter responsible for cadaverine secretion. The biofilm lifecycle of the ΔcadB mutant was identical to that of WT V. cholerae (see Author response image 1), showing that periplasmic/extracellular cadaverine does not alter norspermidine signaling in WT V. cholerae. Moreover, we made the ΔcadB ΔpotD1 double mutant and it behaved identically to the single ΔpotD1 mutant. Thus, the presence or absence of cadaverine does not alter the biofilm dispersal failure phenotype of the ΔpotD1 mutant, so the ΔpotD1 mutant phenotype cannot be due to blocking of porins by cadaverine. Together, our results show that secreted cadaverine does not impinge on the V. cholerae biofilm lifecycle, and therefore, cadaverine has no effect on periplasmic norspermidine levels nor on MbaA activity.

Because our results do not support any role for cadaverine in the V. cholerae biofilm lifecycle, we want to keep the message of our manuscript streamlined. Therefore we have not included an extended analysis of cadaverine in the current manuscript. As noted above, we do include our cadaverine measurements in the Results, and we review information on polyamines in general in the revised Introduction.

Author response image 1

Scholarship issues:

A more thorough scholarly analysis of what is known about NspS (versus proposed here) would bolster the role for NspS in the authors' model. The current manuscript does not describe the key literature on NspS. In particular, the sensing of polyamines by MbaA through NspS-polyamine binding and direct binding of NspS to MbaA seems well-supported by literature that could be described.

We thank the reviewers for this suggestion. We have written a more in-depth passage in the revised Introduction concerning what is known about the MbaA-NspS signaling circuit. Please see lines 78-111.

The paper generally lacks context and does not discuss in adequate detail the general biological roles for polyamines (including cytoplasmically), what's already known about their roles in biofilm formation (in vibrios or other organisms like B. subtilis), or how they are synthesized. Without this sort of context, I think the paper will be difficult to access or appreciate for the non-expert/non-biofilm researcher.

We regret overlooking a basic introduction to polyamines and we agree including such text will broaden the scope of the manuscript. In response, we now describe general polyamine biology in the revised Introduction (lines 69-96).

It is surprising that the authors did not discuss and cite the Sobe et al., paper, "Spermine inhibits Vibrio cholerae biofilm formation through the NspS-Mba polyamine signaling system" (2017) J. Biol. Chem. 292, 17025-17036. This paper discusses the concept of self/non-self mediated by polyamines and makes a persuasive argument that spermine (a primarily eukaryotic polyamine) is a more likely signal than spermidine, especially for a human pathogen.

We thank the reviewer for catching this oversight. We have included a discussion the effect of spermine on V. cholerae biofilm formation in our revised Introduction (lines 85-91).

The authors did not measure cyclic-di-GMP directly and did not measure polyamine transport directly. It would therefore be prudent to make less categorically emphatic statements about eg., diguanylate cyclase activity line 339 "our data show that MbaA does synthesize c-di-GMP when norspermidine is present (Figure 3C, Figure 4C). In fact, c-di-GMP was not measured and the diguanylate cyclase activity of MbaA was not biochemically assayed. Similarly, for norspermidine uptake – in the abstract ("Biofilm dispersal fails in the absence of PotD1 because reuptake of endogenously produced norspermidine does not occur..") and line 109. At this stage, more tentative language would be appropriate.

We thank the reviewer for this comment. We have now toned down our language in these and other cases.

Regarding self/non-self, I had difficulty integrating the discussion proposing that "self" molecules promote biofilm with a role for biofilm dispersal in cholera transmission (when, presumably, the "self" molecules would be at high abundance). I think a more sophisticated discussion of the issue here would be helpful, given that this is the lab that has defined the relevant QS signaling, has distinguished surface biofilms vs. liquid aggregates, etc.

As requested, we have expanded the text on the topic of polyamines as self vs non-self-signals in the Introduction, Results, and Discussion sections, with a focus on comparing the polyamine system to traditional quorum-sensing circuits. For example, see lines 391-408 for our overarching ideas concerning how the NspS-MbaA system could function to distinguish “self” vs “other.” Additionally, see lines 430-444 where we compare and contrast the opposing MbaA enzymatic activities to those of the established quorum-sensing receptor, LuxN. Beyond those new passages, at present, we hesitate to speculate on the role of MbaA signaling in disease transmission absent any data on which to hang our ideas. To our knowledge, NspS-MbaA-mediated signaling has not yet been investigated in animal models of infection.

https://doi.org/10.7554/eLife.65487.sa2

Article and author information

Author details

  1. Andrew A Bridges

    1. Department of Molecular Biology, Princeton University, Princeton, United States
    2. The Howard Hughes Medical Institute, Chevy Chase, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - reviewing and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8132-751X
  2. Bonnie L Bassler

    1. Department of Molecular Biology, Princeton University, Princeton, United States
    2. The Howard Hughes Medical Institute, Chevy Chase, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    bbassler@princeton.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0043-746X

Funding

Howard Hughes Medical Institute

  • Bonnie L Bassler

National Institutes of Health (5R37GM065859)

  • Bonnie L Bassler

National Science Foundation (MCB-1713731)

  • Bonnie L Bassler

Max Planck - Alexander von Humboldt-Stiftung

  • Bonnie L Bassler

Damon Runyon Cancer Research Foundation (DRG-2302-17)

  • Andrew A Bridges

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank members of the Bassler group and Prof. Ned Wingreen for thoughtful discussions. The c-di-GMP reporter plasmid was a kind gift from Fitnat Yildiz (UC Santa Cruz). Mass spectrometry was conducted by the Princeton Molecular Biology Proteomics and Mass Spectrometry Core Facility. This work was supported by the Howard Hughes Medical Institute, NIH Grant 5R37GM065859, National Science Foundation Grant MCB-1713731, and a Max Planck-Alexander von Humboldt research award to BLB. AAB is a Howard Hughes Medical Institute Fellow of the Damon Runyon Cancer Research Foundation, DRG-2302–17. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Senior Editor

  1. Gisela Storz, National Institute of Child Health and Human Development, United States

Reviewing Editor

  1. Michael T Laub, Massachusetts Institute of Technology, United States

Reviewers

  1. Anthony Michael
  2. Mark Mandel

Publication history

  1. Received: December 5, 2020
  2. Accepted: April 13, 2021
  3. Accepted Manuscript published: April 15, 2021 (version 1)
  4. Version of Record published: April 27, 2021 (version 2)

Copyright

© 2021, Bridges and Bassler

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,093
    Page views
  • 201
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Epidemiology and Global Health
    2. Microbiology and Infectious Disease
    Kelsey M Sumner et al.
    Research Article

    Background:Asymptomatic Plasmodium falciparum infections are common in sub-Saharan Africa, but their effect on subsequent symptomaticity is incompletely understood.

    Methods:In a 29-month cohort of 268 people in Western Kenya, we investigated the association between asymptomatic P. falciparum and subsequent symptomatic malaria with frailty Cox models.

    Results:Compared to being uninfected, asymptomatic infections were associated with an increased 1-month likelihood of symptomatic malaria [adjusted Hazard Ratio (aHR):2.61, 95%CI:2.05-3.33], and this association was modified by sex, with females [aHR:3.71, 95%CI:2.62-5.24] at higher risk for symptomaticity than males [aHR:1.76, 95%CI:1.24-2.50]. This increased symptomatic malaria risk was observed for asymptomatic infections of all densities and in people of all ages. Long-term risk was attenuated but still present in children under 5 [29-month aHR:1.38, 95%CI:1.05-1.81].

    Conclusions:In this high-transmission setting, asymptomatic P. falciparum can be quickly followed by symptoms and may be targeted to reduce the incidence of symptomatic illness.

    Funding:This work was supported by the National Institute of Allergy and Infectious Diseases (R21AI126024 to WPO, R01AI146849 to WPO and SMT).

    1. Microbiology and Infectious Disease
    Kelsey A Haugh et al.
    Research Article Updated

    Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous, and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer’s patches to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood-, and milk-borne retroviruses spanning three routes was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue-resident sentinel macrophages for establishing infection.