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Exploiting the commons: cyclic diguanylate regulation of bacterial exopolysaccharide production
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ScienceDirect Exploiting the commons: cyclic diguanylate regulation of bacterial exopolysaccharide production Daniel Pe´rez-Mendoza and Juan Sanjua´n Nowadays, there is increasing interest for bacterial polysaccharides in a wide variety of industrial sectors. This is due to their chemical and reological properties, and also the possibility to be obtained by fermentation processes. Biosynthesis of a growing number of exopolysaccharides (EPS) has been reported to be regulated by the ubiquitous second messenger c-di-GMP in a limited number of bacterial species. Since most bacteria are yet unexplored, it is likely that an unsuspected number and variety of EPS structures activated by c-di-GMP await to be uncovered. In the search of new EPS, manipulation of bacterial c-di-GMP metabolism can be combined with high throughput approaches for screening of large collections of bacteria. In addition, c-di-GMP activation of EPS production and promotion of cell aggregation may have direct applications in environmental industries related with biofuel production or wastewater treatments. Address Dpto. Microbiologı´a del Suelo y Sistemas Simbio´ticos, Estacio´n Experimental del Zaidı´n, CSIC. Prof. Albareda N8 1, 18008 Granada, Spain. Corresponding author: Sanjua´n, Juan ([email protected])
chemical and reological properties. An additional advantage is their stable production under controllable conditions, in contrast to other sources like plants and algae. Among bacterial EPS more widely commercialized are sphingans, xanthan, dextran, alginate and cellulose [1,2]. There is a great variability in the cellular and environmental signals and signalling networks regulating production of bacterial EPS. However, for a growing number of EPS a common feature has emerged during the last decade: regulation by the second messenger c-di-GMP (cyclic diguanylate). c-Di-GMP was discovered by Benziman and coworkers in 1987 as an allosteric regulator of bacterial cellulose synthase and is now considered an ubiquitous second messenger that mediates multiple cellular functions, with a particular involvement in the transition between the bacterial motile and sessile (i.e. biofilm communities) lifestyles. Recent reports have reviewed the targets and mechanisms of c-di-GMP regulation in bacteria [7,8,9]. Here we highlight some of the most noticeable aspects as well as some future prospects for c-di-GMP regulation of EPS production.
Current Opinion in Microbiology 2016, 30:36–43
c-Di-GMP regulated EPS
This review comes from a themed issue on Cell regulation
Up to date, more than ten EPS are known to be regulated by c-di-GMP at some level, including: (i) the widespread bacterial cellulose and poly-b(1–6)-N-acetyl-D-glucosamine (PNAG); (ii) the curdlan, xanthan, mixed-linkage b-glucan (MLG) and unipolar polysaccharide (UPP) produced by plant-associated bacteria; (iii) the Pseudomonads alginate, Psl and Pel; and (iv) other EPS associated with more specific taxa such as the ones recently described in Vibrio (VPS) and Listeria (Table 1). Most of these EPS are activated by c-di-GMP, being xanthan the only known example of an EPS negatively regulated by this second messenger.
Edited by Brice Felden and Ute Ro¨mling
http://dx.doi.org/10.1016/j.mib.2015.12.004 1369-5274/# 2016 Elsevier Ltd. All rights reserved.
Introduction Bacteria can produce a broad array of biopolymers with a variety of chemical structures and composition upon the strain, species and culture conditions [1,2]. Some of these biopolymers are polysaccharides secreted into the surrounding environment, the so-called extracellular polysaccharides or exopolysaccharides (EPS), which often confer a survival advantage by protecting the cell against abiotic and biotic stresses, including host defensive factors. EPS are also main components of the extracellular matrix involved in surface adhesion, cell–cell interactions and formation of biofilms [3–6]. Bacterial biopolymers are gaining increasing interest in the chemistry, food, pharmaceutical and environmental sectors due to their purity, Current Opinion in Microbiology 2016, 30:36–43
Many of the c-di-GMP regulated EPS comprise relatively low structural complexity, such as the linear homopolysaccharides (cellulose, curdlan, MLG, PNAG) or heteropolysaccharides (alginate, VPS), but some are ramified (i.e. Psl, xanthan, Listeria EPS). In some cases composition but not structure are known (Pel, UPP Glucomannan). Among the four general mechanisms for EPS biosynthesis and secretion that have been described (reviewed by [10,11,12]), the c-di-GMP regulated EPS use either synthase-dependent or Wzx/Wzy-dependent pathways. Many of them are produced by synthase dependent www.sciencedirect.com
c-di-GMP regulated EPS Pe´rez-Mendoza and Sanjua´n 37
Table 1 Bacterial exopolysaccharides regulated by c-di-GMP Structure a
EPS
Biosynthesis pathway
Xanthan
Branched heteropolysaccharide: b(1–4)-linked Glc backbone and a side chain of 2 Man and 1 GlcA
Wzx/Wzy dependent
Vibrio EPS (VPS, BRP, CPS)
Linear heteropolysaccharide: Glc (sometimes replaced by GlcNAc), Gal and 2acetamido-2-deoxy-Lguluronic
Wzx/Wzy dependent
Unipolar polysaccharide (UPP)
Heteropolysaccharide: Unsolved structure glucomannan, mainly composed by Glc and Man
Likely Wzx/Wzy dependent (see text)
Psl
Branched heteropolysaccharide: pentasaccharide repeating unit containing D-Man, L-Rha and D-Glc
Wzx/Wzy dependent
Pel
Glc-rich polysaccharide of unknown structure
Unknown
Cellulose
Linear homopolysaccharide: b(1–4)-linked Glc
Synthase dependent
Curdlan
Linear homopolysaccharide: b(1–3)-linked Glc
Synthase dependent
Mixed-linkage b-glucan (MLG)
Linear homopolysaccharide: alternant b(1–4) and b(1–3) linked Glc Linear homopolysaccharide: b(1–6)-linked GlcNAc
Synthase dependent
Poly-b(1–6)-NAcetylglucosamine (PNAG) Listeria EPS
Alginate
Branched heteropolysaccharide: b(1–4)-linked ManNAc decorated with terminal a(1–6)-linked Gal Linear heteropolysaccharide: b(1–4)-linked D-ManA and a-L-GulA
Synthase dependent
Unknown
Synthase dependent
GT b
Regulation by c-di-GMP
Different cytosolic and membrane GTs (Gum M, H, K, I, D, L, F, G) with Inverting and retaining mechanisms and belonging to various CAZY families VpsD and VpsI (GT-B folding and a retaining mechanism belonging to CAZY Family no. 4) VpsK (inverting mechanism belonging to CAZY Family no. 26) Atu1237 (orthologue: RL1662) GT-B folding and a retaining mechanism belonging to CAZY Family no. 4 4 GTs involved: 3 with a GT-B folding and a retaining mechanism belonging to CAZY Family no. 4 (PslF, H and I) and 1 with a GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 (PslC) Cytoplasmic GT PelF with GT-B folding and a retaining mechanism belonging to CAZY Family no. 4 BcsA (orthologues: CelA, AcsA, WssB) with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 CrdS with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 BgsA; with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 PgaC (orthologue: HmsR) with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 PssC, with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 Alg8, with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2
References c
Transcriptional level
[20,45]
Transcriptional level
[21,33,46–53]
Unknown
[54]
Transcriptional level
[15,18,55,56]
Transcriptional and posttranslational level
[15,18,27,57–59]
Transcriptional and posttranslational level
[16,17,29,60–66]
Unknown
[67]
Posttranslational level
[25]
Transcriptional and posttranslational l evel
[14,30,68,69]
Posttranslational level
[13,28]
Posttranslational level
[26,31]
a
Glc, glucose; Gal, galactose; Man, mannose; Rha, rhamnose; GlcA, glucuronic acid; GlcNAc, N-acetyl-glucosamine; ManA, mannuronic acid; GulA, guluronic acid; ManNAc, N-acetyl-mannosamine. Classification of glycosyltransferases (GT) according to CAZy (http://www.cazy.org/GlycosylTransferases.html). c References related with c-di-GMP regulation. b
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Current Opinion in Microbiology 2016, 30:36–43
38 Cell regulation
pathways (Table 1), in which complete polymer strands are secreted across the membranes and the cell wall. The polymerization and translocation process are performed by a single synthase protein, which is often a subunit of an envelope-spanning multiprotein complex [10]. The Psl, xanthan and Vibrio VPS likely utilise Wzx/Wzy-dependent mechanisms (Table 1), where individual glycosidic repeat units are assembled by one or more cytoplasmic glycosyltransferases (GT’s), and linked to a lipid acceptor at the inner membrane. In a next step, a cytoplasmic membrane flipase (Wzx) translocates the lipid-linked oligosaccharide to the periplasmic space where the polymerisation is catalysed by a Wzy protein. The polymer is then exported to the cell surface involving a copolymerase protein (PCP, Wzz) and a member of the outer membrane polysaccharide exporter family (OPX [12]). However, for some c-di-GMP regulated EPS the specific synthesis/secretion pathways remain to be demonstrated (Table 1). The UPP glucomannan could fall within the Wzx/Wzy group since the A. tumefaciens biosynthesis operon encodes proteins with putative Wzy (Atu1235), Wzx (Atu1238) and Wzz (Atu1239) domains. Less clear is the situation regarding Pel [10] and the Listeria EPS [13], whose biosynthetic proteins seem to share properties with synthase-dependent systems. Transcriptional regulation
The alteration of c-di-GMP levels has been reported as having an impact in the transcriptional levels of the cellulose, PNAG, Pel and Psl biosynthetic operons [14– 17]. In contrast to PNAG, where the regulator is not known, the transcriptional regulator involved in the cdi-GMP regulation of Pel and Psl has been extensively studied [18,19]. FleQ, the master regulator of flagella gene expression in Pseudomonas aeruginosa, behaves as a transcriptional repressor via binding to the promoters of pel and psl operons, and this repression is relieved by the direct binding of c-di-GMP to FleQ. A further study revealed that the action of this transcriptional regulator is more complex, showing a dual regulatory role. After cdi-GMP binding, FleQ occupies a different promoter region therefore activating pel operon transcription [19]. In the case of xanthan and VPS, c-di-GMP exerts its action through the binding to a transcriptional regulator and changing its affinity for the promoter of the biosynthetic operons [20,21]. Furthermore, different diguanylate cyclases (DGC) have been described among the genes regulated by the c-di-GMP-dependent transcriptional regulators (VpsT and VpsR) involved in VPS production [22]. This implies that indirect regulation could also be exerted at transcriptional level by c-di-GMP controlling the expression of different bacterial DGCs and phosphodiesterases (PDEs) (Figure 1). Posttranslational regulation of EPS
One of the most extended regulations by c-di-GMP is the posttranslational regulation of the EPS biosynthesis and Current Opinion in Microbiology 2016, 30:36–43
Figure 1
EPS
DGC
GMP Transcriptional Regulator
PDE
GTP
c-di-GMP NDP-
EPS operon NDP
c-di-GMP
DGC or PDE
DGC/PDE GTP/GMP
DGC
GTP
Current Opinion in Microbiology
Regulation of EPS production by c-di-GMP. A Gram-negative bacterial cell is depicted, with cytoplasmic (green line), outer membrane (black line) and periplasmic space (grey). The activities of diguanylate cyclases (DGC) and/or phosphodiesterases (PDE), usually responsive to specific intracellular or external signals, determine c-di-GMP economy. The bacterial second messenger c-di-GMP may regulate exopolysaccharide (EPS) production at two different levels: (i) transcriptional, activating or repressing the expression of the biosynthetic operon or other regulatory genes; (ii) through the allosteric activation of the glycosyl transferase (GT) and/or an accessory protein involved in the polymerisation or secretion of the EPS. A membraneassociated synthase-dependent biosynthetic complex is represented. The c-di-GMP pool involved in activation of the biosynthetic machinery is dependent on the activity of DGCs and/or PDEs, which are often localized near the EPS macromolecular biosynthetic complexes. NDP, activated monosaccharide.
secretion machinery (Figure 1). The positive effects of cdi-GMP via posttranslational regulation have been reported for cellulose, PNAG, alginate, Pel, and recently for MLG in Sinorhizobium meliloti and the Listeria EPS (Table 1). The velocity, flexibility and precise control of cdi-GMP allosteric regulation represent great advantages in the evolution of polysaccharide synthesis and secretion systems [23]. However, even within this specific posttranslational regulation, it is remarkable that different types of cdi-GMP effectors can be involved. One of the best characterised to date is the activation of the cellulose production by direct binding of c-di-GMP to the C-terminal PilZ domain present in cellulose synthase [24]. This activation seems to be mediated by a ‘derepression mechanism’ by which c-di-GMP binding releases an autoinhibited state of the protein, with a so-called ‘gating loop’ that interacts with the PilZ domain and controls access of the substrate to the active site. c-di-GMP binding to the PilZ domain displaces the gating loop from the active site and allows substrate diffusion. Under these conditions, c-di-GMP would not affect Michaelis constant (Km) for the substrate but would increase the fraction of the enzyme that becomes catalytically active [24]. www.sciencedirect.com
c-di-GMP regulated EPS Pe´rez-Mendoza and Sanjua´n 39
Direct binding of c-di-GMP to the glycosyl transferase (GT) BgsA seems also to be involved in the production of a mixed-linkage b(1–4),b(1–3) glucan (MLG) by S. meliloti and likely other species among the rhizobiales [25]. The GT BgsA does not contain a PilZ domain but binds cdi-GMP through the C-terminus of the protein, which may represent a new type of c-di-GMP binding domain. c-di-GMP allosteric activation of alginate can be performed by other PilZ domain. Herein, the PilZ-containing protein is not the GT Alg8 but the accompanying protein Alg44 of the biosynthetic complex. However, the precise mechanism by which alginate synthesis is activated by c-di-GMP-Alg44 is still unclear. Although a derepression mechanism has been suggested, either activation or derepression of Alg8 after c-di-GMP binding to Alg44 are plausible [26]. Other recurrent c-di-GMP effectors involved in EPS biosynthesis are catalytically inactive DGC proteins with degenerate GGDEF domains, which are still able to bind the cyclic dinucleotide (GG[D/E]EF is the proposed conserved signature of active DGCs; [8]). Examples belonging to this group of c-di-GMP effectors are PelD and PssE which are essential for the production of Pel and Listeria EPS, respectively [27,28] (Table 1). To this group of effectors might also belong BcsE, which is required for maximal cellulose production in enterobacteria. BcsE contains another recognized c-di-GMP binding domain, GIL [29], which shares c-di-GMP binding motif and secondary structure with GGDEF domains. The precise mechanisms by which these c-di-GMP effectors exert their action remain elusive. Further complexity and diversity of c-di-GMP effector molecules came with the recent description of the posttranslational regulation of PNAG. In a very elegant approach, Steiner and co-workers described how c-di-GMP regulates PNAG synthesis in E. coli, via the c-di-GMP binding to the interface of the interaction between two proteins required for poly-GlcNAc synthesis, PgaC (the GT) and PgaD. The c-di-GMP binding to the two proteins stabilizes their interaction and promotes enzymatic activity, generating a conformational change in the system that favours the extrusion of the EPS. Furthermore, PgaD is rapidly degraded in the absence of c-di-GMP to switch off the system [30]. EPS production involves highly energy demanding and costly processes and, therefore, their biosynthesis must be tightly regulated. This holds true also for those EPS whose production is regulated by cyclic diguanylate, as often bacteria are equipped to produce more than one c-diGMP regulated EPS. Many of the biosynthetic machineries of c-di-GMP regulated EPS are enzymatic complexes exhibiting an inactive conformation, which is only released at the appropriate time and conditions, through the binding of c-di-GMP to the GT or to an effector interacting with the www.sciencedirect.com
GT (Figure 1). The c-di-GMP signalling ensures a tight control of the biosynthetic process and a rapid transition back to the inactive state when the right environmental conditions disappear. This is reinforced by the fact that the biosynthesis of many of these EPSs are regulated at multiple levels (Table 1). On the other hand, the high diversification and singularity observed in the regulation of the different EPS suggests a rapid evolution to adapt to the specific conditions required in each case. Since c-di-GMP is involved in the regulation of a plethora of different bacterial processes [8], one of the major obstacles that c-di-GMP signalling needs to deal with is target specificity. In the context of posttranslational regulation of the different EPS, c-di-GMP seems to have overcome this issue by co-localizing the c-di-GMP metabolising enzymes (DGC and PDE) close to the EPS macromolecular biosynthetic complexes. For example, MucR regulates alginate production by generating a local c-di-GMP pool in the vicinity of the effector Alg44 [31]. Also direct interactions between the GT HmsR (homologue of PgaC), the DGC HmsT and the PDE HmsP, involved in PNAG synthesis in Yersinia pestis, have been observed, suggesting that HmsR, HmsT and HmsP form an inner membrane enzyme complex [32]. In contrast, a global instead of local signalling may operate during c-diGMP regulation of gene transcription. For instance VpsT, the master regulator of Vibrio EPS synthesis operon expression, does not interact with the DGCs that supply c-di-GMP, suggesting that c-di-GMP from these DGCs diffuses to VpsT, supporting a model of c-di-GMP signalling at a distance [33].
Exploitation of c-di-GMP regulation of EPS Many of the c-di-GMP regulated EPS are cryptic or produced in very low amounts in laboratory cultures, this is, they could be abundantly produced under yet unknown environmental conditions. In several instances EPS discovery was possible after inducing artificial increments of intracellular c-di-GMP in bacteria, leading to deregulation of EPS biosynthesis and enhanced production (Table 1). Considering the limited number of bacterial species and strains explored for c-di-GMP regulated EPS production nowadays, it is very likely that the list of this class of EPS will keep growing in the next years. Besides those listed in Table 1, there is evidence for at least two other, uncharacterized EPS likely regulated by c-di-GMP. In Pseudomonas putida overexpression of the protein Rup4959 which is a transcriptional regulator with DGC activity, enhances the production of a calcofluor stainable EPS of unknown structure [34]. In Burkholderia cenocepacia another c-diGMP responsive transcriptional regulator (Bcam1349) controls production of some biofilm matrix components, including an uncharacterised EPS [35]. Classic approaches for finding new EPS structures and pathways have been motivated by the discovery of their Current Opinion in Microbiology 2016, 30:36–43
40 Cell regulation
biological role (e.g. succinoglycan), or the need to find alternative sources of new or already known polymers with commercial applications (e.g. alginate, cellulose). One major goal of all microbial EPS production systems is to achieve a cost-effective industrial production of the compound, which can be accomplished by overexpressing genes of precursors biosynthesis or EPS synthesis/assembly, sometimes in combination with gene knockouts of pathways competing for precursors (reviewed in [11]). Other strategies are focused on tailor-made variants (i.e., alterations in the degree of polymerization, removal of side chains or non-sugar substituents) and even new EPS structures through genetic engineering and synthetic biology approaches (see e.g. [36]). However, the natural diversity of bacterial EPS is still largely unexplored. Screening of bacterial diversity has often been limited to the search of strains competent in production of a particular EPS. But other approaches and high throughput screening techniques are required to explore the high potential of EPS that likely remain to be discovered [37]. c-di-GMP regulation can be combined with more classic and also with new high throughput approaches and exploited for both, enhancing volumetric productivity as well as finding novel EPS structures and pathways. The widespread activation of bacterial EPSs by c-diGMP can be used as a genetic tool to search for EPS in virtually any bacteria regardless of biological function and structure. As mentioned above, enhancements of intracellular c-di-GMP concentrations very often results in overproduction of c-di-GMP regulated EPS. For this purpose, the recently reported mini-Tn7 vehicles for expression of a highly active DGC can be of broad use [38]. This type of tools can be used to screen a wide range of bacterial species and strains which could lead to uncover an unsuspected number of EPS structures. In addition, it could be combined with genome analyses and protein searches aimed to identify new c-di-GMP regulated EPS synthesis systems, that is, by searching for GTs or related proteins of unknown function containing known c-di-GMP binding domains. An important aspect in the search of novel c-di-GMP regulated EPS is to count with simple methods for detection of EPS-enhanced phenotypes. For several of the known c-di-GMP regulated EPS, detection was facilitated by in vivo staining with the colorants congo red and/or calcofluor. Others, like curdlan, may also be specifically stained with aniline blue. Stains must be nontoxic since they must be able to be incorporated into the growth medium and stain colonies in vivo without affecting viability. Otherwise, colony staining can be done after obtaining colony replicates. Screening is also theoretically possible in case of colorant exclusion or negative staining, for example the case of Sudan Black B exclusion by EPS Current Opinion in Microbiology 2016, 30:36–43
of S. meliloti [39]. Alternatively, screening can be done in liquid cultures by visual observation of viscosity and carbohydrate precipitation, which can be combined with monosaccharide analysis [40]. Enhanced production of one or more EPS by c-di-GMP may have direct biotechnological applications. An increment in EPS production usually correlates with cell aggregation and flocculation, which are important in numerous industrial processes. For instance, recovery of cyanobacteria for biofuel production implies high cost energy actions, such as the use of flocculants, filtration or centrifugation [41]. EPS regulation by c-di-GMP could help to modulate microbial adherence and flocculation properties ‘on demand’ for use in biomass harvest. Removal of pollutants from wastewaters is continuously demanding cheaper and environmentally friendly processes. Adsorption of toxic compounds on polysaccharides is a procedure of choice for treating industrial effluents containing heavy metals or other toxic compounds like dyes from textile industries [42,43]. Cyclic-di-GMP regulation could thus be used in wastewater treatment systems involving either live bacteria overproducing c-di-GMP dependent EPS or EPS crude fractions from such bacteria [44].
Conclusions Bacteria can produce and secrete a broad array of EPS which can serve for various purposes involving the interaction of the cell with its surrounding environment. There is great variability in the cellular and environmental signals regulating the production of bacterial EPS. During the last decade, the production of a growing number of structurally diverse bacterial EPS has been reported to be regulated by the second messenger c-diGMP. Although the molecular mechanisms may vary among different EPS and bacterial species, in most cases EPS biosynthesis is activated by enhanced c-di-GMP intracellular levels. This common regulatory feature can be useful to unravel the largely unexplored diversity of bacteria, to uncover novel EPS structures and biosynthesis pathways. Deregulation of the c-di-GMP economy can be used in combination with classic and modern highthroughput approaches to screen large collections of environmental bacteria for new c-di-GMP regulated EPS. Enhanced production of EPS and cell aggregation by high c-di-GMP can also find direct applications in environmental processes related with biomass production or wastewater treatments.
Acknowledgements Work at the author’s laboratory was supported by grants BIO2011-23032 and BIO2014-55075-P (Ministerio de Economı´a y Competitividad, Spain; cofinanced with FEDER funds), and grant CSIC 201440E026. DPM was supported by Andalucı´a Talent Hub Program launched by the Andalusian Knowledge Agency, co-funded by the European Union’s Seventh Framework Program, Marie Skłodowska-Curie actions (COFUND — Grant Agreement n8 291780) and the Ministry of Economy, Innovation, Science www.sciencedirect.com
c-di-GMP regulated EPS Pe´rez-Mendoza and Sanjua´n 41
and Employment of the Junta de Andalucı´a. A.M. Matia-Gonza´lez is gratefully acknowledged for critical comments on the manuscript.
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32. Bobrov AG, Kirillina O, Forman S, Mack D, Perry RD: Insights into Yersinia pestis biofilm development: topology and cointeraction of Hms inner membrane proteins involved in exopolysaccharide production. Environ Microbiol 2008, 10:1419-1432. 33. Shikuma NJ, Fong JC, Yildiz FH: Cellular levels and binding of cdi-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT. PLoS Pathog 2012, 8:e1002719. 34. Matilla MA, Travieso ML, Ramos JL, Ramos-Gonzalez MI: Cyclic diguanylate turnover mediated by the sole GGDEF/EAL response regulator in Pseudomonas putida: its role in the rhizosphere and an analysis of its target processes. Environ Microbiol 2011, 13:1745-1766. 35. Fazli M, McCarthy Y, Givskov M, Ryan RP, Tolker-Nielsen T: The exopolysaccharide gene cluster Bcam1330-Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349. Microbiol Open 2013, 2:105-122. 36. Becker A: Challenges and perspectives in combinatorial assembly of novel exopolysaccharide biosynthesis pathways. Front Microbiol 2015, 6:687. 37. Ru¨hmann B, Schmid J, Sieber V: Methods to identify the unexplored diversity of microbial exopolysaccharides. Front Microbiol 2015, 6:565. 38. Romero-Jime´nez L, Rodrı´guez-Carbonell D, Gallegos MT, Sanjua´n J, Pe´rez-Mendoza D: Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria. BMC Microbiol 2015, 15:190. Set of mini-Tn7 vehicles to allow integration and stable expression of a DGC in bacteria. Artificial increases the c-di-GMP contents is a useful tool to enhancing volumetric productivity of EPS as well as finding novel EPS. 39. Liu M, Gonzalez JE, Willis LB, Walker GC: A novel screening method for isolating exopolysaccharide-deficient mutants. Appl Environ Microbiol 1998, 64:4600-4602. 40. Ru¨hmann B, Schmid J, Sieber V: High throughput exopolysaccharide screening platform: from strain cultivation to monosaccharide composition and carbohydrate fingerprinting in one day. Carbohydr Polym 2015, 122:212-220. 41. Jittawuttipoka T, Planchon M, Spalla O, Benzerara K, Guyot F, Cassier-Chauvat C, Chauvat F: Multidisciplinary evidences that Synechocystis PCC6803 exopolysaccharides operate in cell sedimentation and protection against salt and metal stresses. PLoS One 2013, 8:e55564. 42. Blackburn RS: Natural polysaccharides and their interactions with dye molecules: applications in effluent treatment. Environ Sci Technol 2004, 38:4905-4909.
49. Waters CM, Lu W, Rabinowitz JD, Bassler BL: Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cyclic di-GMP levels and repression of vpsT. J Bacteriol 2008, 190:2527-2536. 50. Srivastava D, Harris RC, Waters CM: Integration of cyclic diGMP and quorum sensing in the control of vpsT and aphA in Vibrio cholerae. J Bacteriol 2011, 193:6331-6341. 51. Guo Y, Rowe-Magnus DA: Identification of a c-di-GMPregulated polysaccharide locus governing stress resistance and biofilm and rugose colony formation in Vibrio vulnificus. Infect Immun 2010, 78:1390-1402. 52. Garrison-Schilling KL, Kaluskar ZM, Lambert B, Pettis GS: Genetic analysis and prevalence studies of the brp exopolysaccharide locus of Vibrio vulnificus. PLoS One 2014, 9:e100890. 53. Ferreira RBR, Chodur DM, Antunes LCM, Trimble MJ, McCarter LL: Output targets and transcriptional regulation by a cyclic dimeric GMP-responsive circuit in the Vibrio parahaemolyticus Scr network. J Bacteriol 2012, 194:914-924. 54. Xu J, Kim J, Koestler BJ, Choi JH, Waters CM, Fuqua C: Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile-to-sessile switch. Mol Microbiol 2013, 89:929-948. 55. Irie Y, Borlee BR, O’Connor JR, Hill PJ, Harwood CS, Wozniak DJ, Parsek MR: Self-produced exopolysaccharide is a signal that stimulates biofilm formation in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2012, 109:20632-20636. 56. Byrd MS, Sadovskaya I, Vinogradov E, Lu H, Sprinkle AB, Richardson SH, Ma L, Ralston B, Parsek MR, Anderson EM et al.: Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Mol Microbiol 2009, 73:622-638. 57. Merritt JH, Ha D-G, Cowles KN, Lu W, Morales DK, Rabinowitz J, Gitai Z, O’Toole GA: Specific control of Pseudomonas aeruginosa surface-associated behaviors by two c-di-GMP diguanylate cyclases. mBio 2010, 1 e00183-00110. 58. Malone JG, Jaeger T, Spangler C, Ritz D, Spang A, Arrieumerlou C, Kaever V, Landmann R, Jenal U: YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa. PLoS Pathog 2010, 6:e1000804. 59. Arago´n IM, Pe´rez-Mendoza D, Gallegos MT, Ramos C: The c-diGMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex. Mol Plant Pathol 2014, 16:604-615.
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Current Opinion in Microbiology 2016, 30:36–43
ScienceDirect Exploiting the commons: cyclic diguanylate regulation of bacterial exopolysaccharide production Daniel Pe´rez-Mendoza and Juan Sanjua´n Nowadays, there is increasing interest for bacterial polysaccharides in a wide variety of industrial sectors. This is due to their chemical and reological properties, and also the possibility to be obtained by fermentation processes. Biosynthesis of a growing number of exopolysaccharides (EPS) has been reported to be regulated by the ubiquitous second messenger c-di-GMP in a limited number of bacterial species. Since most bacteria are yet unexplored, it is likely that an unsuspected number and variety of EPS structures activated by c-di-GMP await to be uncovered. In the search of new EPS, manipulation of bacterial c-di-GMP metabolism can be combined with high throughput approaches for screening of large collections of bacteria. In addition, c-di-GMP activation of EPS production and promotion of cell aggregation may have direct applications in environmental industries related with biofuel production or wastewater treatments. Address Dpto. Microbiologı´a del Suelo y Sistemas Simbio´ticos, Estacio´n Experimental del Zaidı´n, CSIC. Prof. Albareda N8 1, 18008 Granada, Spain. Corresponding author: Sanjua´n, Juan ([email protected])
chemical and reological properties. An additional advantage is their stable production under controllable conditions, in contrast to other sources like plants and algae. Among bacterial EPS more widely commercialized are sphingans, xanthan, dextran, alginate and cellulose [1,2]. There is a great variability in the cellular and environmental signals and signalling networks regulating production of bacterial EPS. However, for a growing number of EPS a common feature has emerged during the last decade: regulation by the second messenger c-di-GMP (cyclic diguanylate). c-Di-GMP was discovered by Benziman and coworkers in 1987 as an allosteric regulator of bacterial cellulose synthase and is now considered an ubiquitous second messenger that mediates multiple cellular functions, with a particular involvement in the transition between the bacterial motile and sessile (i.e. biofilm communities) lifestyles. Recent reports have reviewed the targets and mechanisms of c-di-GMP regulation in bacteria [7,8,9]. Here we highlight some of the most noticeable aspects as well as some future prospects for c-di-GMP regulation of EPS production.
Current Opinion in Microbiology 2016, 30:36–43
c-Di-GMP regulated EPS
This review comes from a themed issue on Cell regulation
Up to date, more than ten EPS are known to be regulated by c-di-GMP at some level, including: (i) the widespread bacterial cellulose and poly-b(1–6)-N-acetyl-D-glucosamine (PNAG); (ii) the curdlan, xanthan, mixed-linkage b-glucan (MLG) and unipolar polysaccharide (UPP) produced by plant-associated bacteria; (iii) the Pseudomonads alginate, Psl and Pel; and (iv) other EPS associated with more specific taxa such as the ones recently described in Vibrio (VPS) and Listeria (Table 1). Most of these EPS are activated by c-di-GMP, being xanthan the only known example of an EPS negatively regulated by this second messenger.
Edited by Brice Felden and Ute Ro¨mling
http://dx.doi.org/10.1016/j.mib.2015.12.004 1369-5274/# 2016 Elsevier Ltd. All rights reserved.
Introduction Bacteria can produce a broad array of biopolymers with a variety of chemical structures and composition upon the strain, species and culture conditions [1,2]. Some of these biopolymers are polysaccharides secreted into the surrounding environment, the so-called extracellular polysaccharides or exopolysaccharides (EPS), which often confer a survival advantage by protecting the cell against abiotic and biotic stresses, including host defensive factors. EPS are also main components of the extracellular matrix involved in surface adhesion, cell–cell interactions and formation of biofilms [3–6]. Bacterial biopolymers are gaining increasing interest in the chemistry, food, pharmaceutical and environmental sectors due to their purity, Current Opinion in Microbiology 2016, 30:36–43
Many of the c-di-GMP regulated EPS comprise relatively low structural complexity, such as the linear homopolysaccharides (cellulose, curdlan, MLG, PNAG) or heteropolysaccharides (alginate, VPS), but some are ramified (i.e. Psl, xanthan, Listeria EPS). In some cases composition but not structure are known (Pel, UPP Glucomannan). Among the four general mechanisms for EPS biosynthesis and secretion that have been described (reviewed by [10,11,12]), the c-di-GMP regulated EPS use either synthase-dependent or Wzx/Wzy-dependent pathways. Many of them are produced by synthase dependent www.sciencedirect.com
c-di-GMP regulated EPS Pe´rez-Mendoza and Sanjua´n 37
Table 1 Bacterial exopolysaccharides regulated by c-di-GMP Structure a
EPS
Biosynthesis pathway
Xanthan
Branched heteropolysaccharide: b(1–4)-linked Glc backbone and a side chain of 2 Man and 1 GlcA
Wzx/Wzy dependent
Vibrio EPS (VPS, BRP, CPS)
Linear heteropolysaccharide: Glc (sometimes replaced by GlcNAc), Gal and 2acetamido-2-deoxy-Lguluronic
Wzx/Wzy dependent
Unipolar polysaccharide (UPP)
Heteropolysaccharide: Unsolved structure glucomannan, mainly composed by Glc and Man
Likely Wzx/Wzy dependent (see text)
Psl
Branched heteropolysaccharide: pentasaccharide repeating unit containing D-Man, L-Rha and D-Glc
Wzx/Wzy dependent
Pel
Glc-rich polysaccharide of unknown structure
Unknown
Cellulose
Linear homopolysaccharide: b(1–4)-linked Glc
Synthase dependent
Curdlan
Linear homopolysaccharide: b(1–3)-linked Glc
Synthase dependent
Mixed-linkage b-glucan (MLG)
Linear homopolysaccharide: alternant b(1–4) and b(1–3) linked Glc Linear homopolysaccharide: b(1–6)-linked GlcNAc
Synthase dependent
Poly-b(1–6)-NAcetylglucosamine (PNAG) Listeria EPS
Alginate
Branched heteropolysaccharide: b(1–4)-linked ManNAc decorated with terminal a(1–6)-linked Gal Linear heteropolysaccharide: b(1–4)-linked D-ManA and a-L-GulA
Synthase dependent
Unknown
Synthase dependent
GT b
Regulation by c-di-GMP
Different cytosolic and membrane GTs (Gum M, H, K, I, D, L, F, G) with Inverting and retaining mechanisms and belonging to various CAZY families VpsD and VpsI (GT-B folding and a retaining mechanism belonging to CAZY Family no. 4) VpsK (inverting mechanism belonging to CAZY Family no. 26) Atu1237 (orthologue: RL1662) GT-B folding and a retaining mechanism belonging to CAZY Family no. 4 4 GTs involved: 3 with a GT-B folding and a retaining mechanism belonging to CAZY Family no. 4 (PslF, H and I) and 1 with a GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 (PslC) Cytoplasmic GT PelF with GT-B folding and a retaining mechanism belonging to CAZY Family no. 4 BcsA (orthologues: CelA, AcsA, WssB) with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 CrdS with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 BgsA; with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 PgaC (orthologue: HmsR) with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 PssC, with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2 Alg8, with GT-A folding and an inverting mechanism belonging to CAZY Family no. 2
References c
Transcriptional level
[20,45]
Transcriptional level
[21,33,46–53]
Unknown
[54]
Transcriptional level
[15,18,55,56]
Transcriptional and posttranslational level
[15,18,27,57–59]
Transcriptional and posttranslational level
[16,17,29,60–66]
Unknown
[67]
Posttranslational level
[25]
Transcriptional and posttranslational l evel
[14,30,68,69]
Posttranslational level
[13,28]
Posttranslational level
[26,31]
a
Glc, glucose; Gal, galactose; Man, mannose; Rha, rhamnose; GlcA, glucuronic acid; GlcNAc, N-acetyl-glucosamine; ManA, mannuronic acid; GulA, guluronic acid; ManNAc, N-acetyl-mannosamine. Classification of glycosyltransferases (GT) according to CAZy (http://www.cazy.org/GlycosylTransferases.html). c References related with c-di-GMP regulation. b
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Current Opinion in Microbiology 2016, 30:36–43
38 Cell regulation
pathways (Table 1), in which complete polymer strands are secreted across the membranes and the cell wall. The polymerization and translocation process are performed by a single synthase protein, which is often a subunit of an envelope-spanning multiprotein complex [10]. The Psl, xanthan and Vibrio VPS likely utilise Wzx/Wzy-dependent mechanisms (Table 1), where individual glycosidic repeat units are assembled by one or more cytoplasmic glycosyltransferases (GT’s), and linked to a lipid acceptor at the inner membrane. In a next step, a cytoplasmic membrane flipase (Wzx) translocates the lipid-linked oligosaccharide to the periplasmic space where the polymerisation is catalysed by a Wzy protein. The polymer is then exported to the cell surface involving a copolymerase protein (PCP, Wzz) and a member of the outer membrane polysaccharide exporter family (OPX [12]). However, for some c-di-GMP regulated EPS the specific synthesis/secretion pathways remain to be demonstrated (Table 1). The UPP glucomannan could fall within the Wzx/Wzy group since the A. tumefaciens biosynthesis operon encodes proteins with putative Wzy (Atu1235), Wzx (Atu1238) and Wzz (Atu1239) domains. Less clear is the situation regarding Pel [10] and the Listeria EPS [13], whose biosynthetic proteins seem to share properties with synthase-dependent systems. Transcriptional regulation
The alteration of c-di-GMP levels has been reported as having an impact in the transcriptional levels of the cellulose, PNAG, Pel and Psl biosynthetic operons [14– 17]. In contrast to PNAG, where the regulator is not known, the transcriptional regulator involved in the cdi-GMP regulation of Pel and Psl has been extensively studied [18,19]. FleQ, the master regulator of flagella gene expression in Pseudomonas aeruginosa, behaves as a transcriptional repressor via binding to the promoters of pel and psl operons, and this repression is relieved by the direct binding of c-di-GMP to FleQ. A further study revealed that the action of this transcriptional regulator is more complex, showing a dual regulatory role. After cdi-GMP binding, FleQ occupies a different promoter region therefore activating pel operon transcription [19]. In the case of xanthan and VPS, c-di-GMP exerts its action through the binding to a transcriptional regulator and changing its affinity for the promoter of the biosynthetic operons [20,21]. Furthermore, different diguanylate cyclases (DGC) have been described among the genes regulated by the c-di-GMP-dependent transcriptional regulators (VpsT and VpsR) involved in VPS production [22]. This implies that indirect regulation could also be exerted at transcriptional level by c-di-GMP controlling the expression of different bacterial DGCs and phosphodiesterases (PDEs) (Figure 1). Posttranslational regulation of EPS
One of the most extended regulations by c-di-GMP is the posttranslational regulation of the EPS biosynthesis and Current Opinion in Microbiology 2016, 30:36–43
Figure 1
EPS
DGC
GMP Transcriptional Regulator
PDE
GTP
c-di-GMP NDP-
EPS operon NDP
c-di-GMP
DGC or PDE
DGC/PDE GTP/GMP
DGC
GTP
Current Opinion in Microbiology
Regulation of EPS production by c-di-GMP. A Gram-negative bacterial cell is depicted, with cytoplasmic (green line), outer membrane (black line) and periplasmic space (grey). The activities of diguanylate cyclases (DGC) and/or phosphodiesterases (PDE), usually responsive to specific intracellular or external signals, determine c-di-GMP economy. The bacterial second messenger c-di-GMP may regulate exopolysaccharide (EPS) production at two different levels: (i) transcriptional, activating or repressing the expression of the biosynthetic operon or other regulatory genes; (ii) through the allosteric activation of the glycosyl transferase (GT) and/or an accessory protein involved in the polymerisation or secretion of the EPS. A membraneassociated synthase-dependent biosynthetic complex is represented. The c-di-GMP pool involved in activation of the biosynthetic machinery is dependent on the activity of DGCs and/or PDEs, which are often localized near the EPS macromolecular biosynthetic complexes. NDP, activated monosaccharide.
secretion machinery (Figure 1). The positive effects of cdi-GMP via posttranslational regulation have been reported for cellulose, PNAG, alginate, Pel, and recently for MLG in Sinorhizobium meliloti and the Listeria EPS (Table 1). The velocity, flexibility and precise control of cdi-GMP allosteric regulation represent great advantages in the evolution of polysaccharide synthesis and secretion systems [23]. However, even within this specific posttranslational regulation, it is remarkable that different types of cdi-GMP effectors can be involved. One of the best characterised to date is the activation of the cellulose production by direct binding of c-di-GMP to the C-terminal PilZ domain present in cellulose synthase [24]. This activation seems to be mediated by a ‘derepression mechanism’ by which c-di-GMP binding releases an autoinhibited state of the protein, with a so-called ‘gating loop’ that interacts with the PilZ domain and controls access of the substrate to the active site. c-di-GMP binding to the PilZ domain displaces the gating loop from the active site and allows substrate diffusion. Under these conditions, c-di-GMP would not affect Michaelis constant (Km) for the substrate but would increase the fraction of the enzyme that becomes catalytically active [24]. www.sciencedirect.com
c-di-GMP regulated EPS Pe´rez-Mendoza and Sanjua´n 39
Direct binding of c-di-GMP to the glycosyl transferase (GT) BgsA seems also to be involved in the production of a mixed-linkage b(1–4),b(1–3) glucan (MLG) by S. meliloti and likely other species among the rhizobiales [25]. The GT BgsA does not contain a PilZ domain but binds cdi-GMP through the C-terminus of the protein, which may represent a new type of c-di-GMP binding domain. c-di-GMP allosteric activation of alginate can be performed by other PilZ domain. Herein, the PilZ-containing protein is not the GT Alg8 but the accompanying protein Alg44 of the biosynthetic complex. However, the precise mechanism by which alginate synthesis is activated by c-di-GMP-Alg44 is still unclear. Although a derepression mechanism has been suggested, either activation or derepression of Alg8 after c-di-GMP binding to Alg44 are plausible [26]. Other recurrent c-di-GMP effectors involved in EPS biosynthesis are catalytically inactive DGC proteins with degenerate GGDEF domains, which are still able to bind the cyclic dinucleotide (GG[D/E]EF is the proposed conserved signature of active DGCs; [8]). Examples belonging to this group of c-di-GMP effectors are PelD and PssE which are essential for the production of Pel and Listeria EPS, respectively [27,28] (Table 1). To this group of effectors might also belong BcsE, which is required for maximal cellulose production in enterobacteria. BcsE contains another recognized c-di-GMP binding domain, GIL [29], which shares c-di-GMP binding motif and secondary structure with GGDEF domains. The precise mechanisms by which these c-di-GMP effectors exert their action remain elusive. Further complexity and diversity of c-di-GMP effector molecules came with the recent description of the posttranslational regulation of PNAG. In a very elegant approach, Steiner and co-workers described how c-di-GMP regulates PNAG synthesis in E. coli, via the c-di-GMP binding to the interface of the interaction between two proteins required for poly-GlcNAc synthesis, PgaC (the GT) and PgaD. The c-di-GMP binding to the two proteins stabilizes their interaction and promotes enzymatic activity, generating a conformational change in the system that favours the extrusion of the EPS. Furthermore, PgaD is rapidly degraded in the absence of c-di-GMP to switch off the system [30]. EPS production involves highly energy demanding and costly processes and, therefore, their biosynthesis must be tightly regulated. This holds true also for those EPS whose production is regulated by cyclic diguanylate, as often bacteria are equipped to produce more than one c-diGMP regulated EPS. Many of the biosynthetic machineries of c-di-GMP regulated EPS are enzymatic complexes exhibiting an inactive conformation, which is only released at the appropriate time and conditions, through the binding of c-di-GMP to the GT or to an effector interacting with the www.sciencedirect.com
GT (Figure 1). The c-di-GMP signalling ensures a tight control of the biosynthetic process and a rapid transition back to the inactive state when the right environmental conditions disappear. This is reinforced by the fact that the biosynthesis of many of these EPSs are regulated at multiple levels (Table 1). On the other hand, the high diversification and singularity observed in the regulation of the different EPS suggests a rapid evolution to adapt to the specific conditions required in each case. Since c-di-GMP is involved in the regulation of a plethora of different bacterial processes [8], one of the major obstacles that c-di-GMP signalling needs to deal with is target specificity. In the context of posttranslational regulation of the different EPS, c-di-GMP seems to have overcome this issue by co-localizing the c-di-GMP metabolising enzymes (DGC and PDE) close to the EPS macromolecular biosynthetic complexes. For example, MucR regulates alginate production by generating a local c-di-GMP pool in the vicinity of the effector Alg44 [31]. Also direct interactions between the GT HmsR (homologue of PgaC), the DGC HmsT and the PDE HmsP, involved in PNAG synthesis in Yersinia pestis, have been observed, suggesting that HmsR, HmsT and HmsP form an inner membrane enzyme complex [32]. In contrast, a global instead of local signalling may operate during c-diGMP regulation of gene transcription. For instance VpsT, the master regulator of Vibrio EPS synthesis operon expression, does not interact with the DGCs that supply c-di-GMP, suggesting that c-di-GMP from these DGCs diffuses to VpsT, supporting a model of c-di-GMP signalling at a distance [33].
Exploitation of c-di-GMP regulation of EPS Many of the c-di-GMP regulated EPS are cryptic or produced in very low amounts in laboratory cultures, this is, they could be abundantly produced under yet unknown environmental conditions. In several instances EPS discovery was possible after inducing artificial increments of intracellular c-di-GMP in bacteria, leading to deregulation of EPS biosynthesis and enhanced production (Table 1). Considering the limited number of bacterial species and strains explored for c-di-GMP regulated EPS production nowadays, it is very likely that the list of this class of EPS will keep growing in the next years. Besides those listed in Table 1, there is evidence for at least two other, uncharacterized EPS likely regulated by c-di-GMP. In Pseudomonas putida overexpression of the protein Rup4959 which is a transcriptional regulator with DGC activity, enhances the production of a calcofluor stainable EPS of unknown structure [34]. In Burkholderia cenocepacia another c-diGMP responsive transcriptional regulator (Bcam1349) controls production of some biofilm matrix components, including an uncharacterised EPS [35]. Classic approaches for finding new EPS structures and pathways have been motivated by the discovery of their Current Opinion in Microbiology 2016, 30:36–43
40 Cell regulation
biological role (e.g. succinoglycan), or the need to find alternative sources of new or already known polymers with commercial applications (e.g. alginate, cellulose). One major goal of all microbial EPS production systems is to achieve a cost-effective industrial production of the compound, which can be accomplished by overexpressing genes of precursors biosynthesis or EPS synthesis/assembly, sometimes in combination with gene knockouts of pathways competing for precursors (reviewed in [11]). Other strategies are focused on tailor-made variants (i.e., alterations in the degree of polymerization, removal of side chains or non-sugar substituents) and even new EPS structures through genetic engineering and synthetic biology approaches (see e.g. [36]). However, the natural diversity of bacterial EPS is still largely unexplored. Screening of bacterial diversity has often been limited to the search of strains competent in production of a particular EPS. But other approaches and high throughput screening techniques are required to explore the high potential of EPS that likely remain to be discovered [37]. c-di-GMP regulation can be combined with more classic and also with new high throughput approaches and exploited for both, enhancing volumetric productivity as well as finding novel EPS structures and pathways. The widespread activation of bacterial EPSs by c-diGMP can be used as a genetic tool to search for EPS in virtually any bacteria regardless of biological function and structure. As mentioned above, enhancements of intracellular c-di-GMP concentrations very often results in overproduction of c-di-GMP regulated EPS. For this purpose, the recently reported mini-Tn7 vehicles for expression of a highly active DGC can be of broad use [38]. This type of tools can be used to screen a wide range of bacterial species and strains which could lead to uncover an unsuspected number of EPS structures. In addition, it could be combined with genome analyses and protein searches aimed to identify new c-di-GMP regulated EPS synthesis systems, that is, by searching for GTs or related proteins of unknown function containing known c-di-GMP binding domains. An important aspect in the search of novel c-di-GMP regulated EPS is to count with simple methods for detection of EPS-enhanced phenotypes. For several of the known c-di-GMP regulated EPS, detection was facilitated by in vivo staining with the colorants congo red and/or calcofluor. Others, like curdlan, may also be specifically stained with aniline blue. Stains must be nontoxic since they must be able to be incorporated into the growth medium and stain colonies in vivo without affecting viability. Otherwise, colony staining can be done after obtaining colony replicates. Screening is also theoretically possible in case of colorant exclusion or negative staining, for example the case of Sudan Black B exclusion by EPS Current Opinion in Microbiology 2016, 30:36–43
of S. meliloti [39]. Alternatively, screening can be done in liquid cultures by visual observation of viscosity and carbohydrate precipitation, which can be combined with monosaccharide analysis [40]. Enhanced production of one or more EPS by c-di-GMP may have direct biotechnological applications. An increment in EPS production usually correlates with cell aggregation and flocculation, which are important in numerous industrial processes. For instance, recovery of cyanobacteria for biofuel production implies high cost energy actions, such as the use of flocculants, filtration or centrifugation [41]. EPS regulation by c-di-GMP could help to modulate microbial adherence and flocculation properties ‘on demand’ for use in biomass harvest. Removal of pollutants from wastewaters is continuously demanding cheaper and environmentally friendly processes. Adsorption of toxic compounds on polysaccharides is a procedure of choice for treating industrial effluents containing heavy metals or other toxic compounds like dyes from textile industries [42,43]. Cyclic-di-GMP regulation could thus be used in wastewater treatment systems involving either live bacteria overproducing c-di-GMP dependent EPS or EPS crude fractions from such bacteria [44].
Conclusions Bacteria can produce and secrete a broad array of EPS which can serve for various purposes involving the interaction of the cell with its surrounding environment. There is great variability in the cellular and environmental signals regulating the production of bacterial EPS. During the last decade, the production of a growing number of structurally diverse bacterial EPS has been reported to be regulated by the second messenger c-diGMP. Although the molecular mechanisms may vary among different EPS and bacterial species, in most cases EPS biosynthesis is activated by enhanced c-di-GMP intracellular levels. This common regulatory feature can be useful to unravel the largely unexplored diversity of bacteria, to uncover novel EPS structures and biosynthesis pathways. Deregulation of the c-di-GMP economy can be used in combination with classic and modern highthroughput approaches to screen large collections of environmental bacteria for new c-di-GMP regulated EPS. Enhanced production of EPS and cell aggregation by high c-di-GMP can also find direct applications in environmental processes related with biomass production or wastewater treatments.
Acknowledgements Work at the author’s laboratory was supported by grants BIO2011-23032 and BIO2014-55075-P (Ministerio de Economı´a y Competitividad, Spain; cofinanced with FEDER funds), and grant CSIC 201440E026. DPM was supported by Andalucı´a Talent Hub Program launched by the Andalusian Knowledge Agency, co-funded by the European Union’s Seventh Framework Program, Marie Skłodowska-Curie actions (COFUND — Grant Agreement n8 291780) and the Ministry of Economy, Innovation, Science www.sciencedirect.com
c-di-GMP regulated EPS Pe´rez-Mendoza and Sanjua´n 41
and Employment of the Junta de Andalucı´a. A.M. Matia-Gonza´lez is gratefully acknowledged for critical comments on the manuscript.
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