Abstract
Certain cyanobacteria can form symbiotic associations with plants, where the symbiont supplies the plant partner with nitrogen and in return obtains sugars. We recently showed that in the symbiotic cyanobacterium Nostoc punctiforme, a glucose specific permease, GlcP, is necessary for the symbiosis to be formed. Results presented here from growth yield measurements of mutant strains with inactivated or overexpressing sugar transporters suggest that GlcP could be induced by a symbiosis specific substance. We also discuss that the transporter may have a role other than nutritional once the symbiosis is established, i.e., during infection, and more specifically in the chemotaxis of the symbiont. Phylogenetic analysis shows that the distribution of GlcP among cyanobacteria is likely influenced by horizontal gene transfer, but also that it is not correlated with symbiotic competence. Instead, regulatory patterns of the transporter in Nostoc punctiforme likely constitute symbiosis specific adaptations.
Keywords: Cyanobacteria, Symbiosis, Nostoc punctiforme, Anthoceros punctatus, Sugar transporter, GlcP, Chemotaxis, Phylogeny
While most cyanobacteria are photoautotrophs and use ammonium or nitrate as a source of nitrogen, other metabolic capacities are not uncommon within this group of organisms. The symbiotic cyanobacterium Nostoc punctiforme is able to use N2 as the nitrogen source and to grow heterotrophically using sugars as carbon source.1 In plant-cyanobacteria symbiosis, the cyanobacterium, which has low photosynthetic activity, provides the plant with fixed nitrogen, while the plant supplies the cyanobacterium with sugars.2
We recently characterized a glucose-specific permease (GlcP) as being necessary for the cyanobacterium N. punctiforme to form symbiosis with the plant Anthoceros punctatus.3 The key observation of our work was that mutants of N. punctiforme in which glucose uptake was abolished did not infect A. punctatus. The gene encoding GlcP is part of a cluster, frtA1-frtA2-frtB-frtC-glcP—oprB, in which at least the frt genes and glcP may be co-transcribed. The frt genes encode an ABC-type transporter for fructose, and oprB encodes an outer membrane sugar porin. We did not determine, however, the exact role of GlcP in symbiosis or its regulation. Possibly the transporter has a role in the mature symbiosis, by taking up sugars supplied by the plant for nutrition, but a second role could be in chemotaxis.
Glucose-Supported Growth
When grown in light the growth yield of the N. punctiforme wild type increased upon the addition of either fructose or glucose (Fig. 1). In contrast, mutant strain CSME1A, which exhibits minimal transport activity of both glucose and fructose,3 showed no growth increase in response to sugar addition, and strain CSME11, which has a low glucose transport activity but retains a partial activity of fructose transport,3 showed a limited increase in growth yield specifically with fructose. On the other hand, glucose increased the yield of the strain overexpressing glcP (CSME1B). These results indicate that in free-living cultures of N. punctiforme GlcP is not expressed at levels permitting the maximal growth that would be possible with glucose, and likely that GlcP is not induced by this sugar. The Frt transporter is neither significantly induced by fructose in N. punctiforme (JC Meeks, personal communication). In contrast, the Frt transporters of the non-symbiotic cyanobacteria Anabaena variabilis and Nostoc sp. strain PCC 7107 are induced by fructose.4,5 The regulation of the sugar transporters GlcP and Frt in N. punctiforme is probably adapted to symbiosis. The transporters could be regulated by a symbiosis-specific mechanism (via a plant derived molecule different from glucose), which may include the nearby located hrm genes that are involved in hormogonia differentiation.6
Figure 1. Response of Nostoc punctiforme to sugars. Yields of cultures of Nostoc punctiforme (orange bars) and mutants CSME1A (Δfrt::C.K3; gene cassette in opposite orientation to the frt-glcP operon) (red bars), CSME11 (ΔglcP-ΔoprB::C.K3) (blue bars), and CSME1B (Δfrt::C.K3; gene cassette in the same orientation as the frt-glcP operon, promoting overexpression of glcP) (black bars) after growth in liquid BG11 (nitrate-containing) medium for 10 d at 30 °C in the light (about 25 µmol m−2 s−1), in the absence or presence of 10 mM fructose or glucose as indicated. See Ekman et al. (2013) for a detailed description of the mutants.
Does GlcP have a Role in Chemotaxis?
Previous studies have shown that inactivation of various genes involved in N2 fixation results in N. punctiforme still forming colonies in the plant partner that are however unable to provide the plant with fixed nitrogen.6 This is in contrast to the glcP mutants, which formed no visible symbiotic colonies, implying that GlcP has a role during the establishment of the symbiosis. The fact that inactivation of glcP does not impair hormogonia differentiation or motility may imply that the early role of GlcP is related to chemotaxis. Indeed, Nostoc is known to be chemotactic toward glucose but not fructose,7 and N-stressed glands of Gunnera plants are enriched in soluble sugars prior to symbiosis.8 Genes encoding homologs of E. coli methyl-accepting chemotaxis proteins (MCPs) are present in N. punctiforme, but gene inactivation studies have failed to identify any of these MCPs as involved in the symbiotic chemotaxis response (JC Meeks, personal communication). GlcP belongs to the Major Facilitator Superfamily (MFS) type of transporters. Chemotaxis dependent on MFS transporters was previously shown for Pseudomonas putida and Ralstonia eutropha.9,10 In both organisms, the transporter-encoding genes cluster together with genes encoding metabolic enzymes of the transported compound, similar to the hrm genes that are located immediately upstream of the frt-glcP genes in N. punctiforme.6 This could suggest the involvement of a metabolic intermediate as an intracellular signal, i.e., metabolic-dependent chemotaxis.
Evolutionary Considerations
To further investigate the biological context of GlcP, we searched for homologs in other bacteria, including 4 symbiotic cyanobacteria whose genomes were recently sequenced, the symbiont of Azolla, Nostoc azollae 0708, 2 symbionts of diatoms, Richelia intracellularis and Calothrix rhizosoleniae SC01, and the symbiont of a prymnesiophyte, UCYN-A.11-13 A Blast search against the nr database showed that the top 12 scoring sequences, with 64–84% sequence identity, were all cyanobacterial (Table 1), while 4 other cyanobacterial sequences showed 53–55% sequence identity. Remaining cyanobacterial hits had 20–30% sequence identity. Hits for the 4 symbiotic cyanobacteria were, if at all present, found in this low scoring group (Table 1). It thus appears that the presence or absence of GlcP is not by itself related to symbiotic competence, and that sugar uptake mechanisms are diverse in cyanobacterial symbionts. Indeed, a glucose transporter different from GlcP was recently characterized in marine picocyanobacteria.14
Table 1. Cyanobacteria with GlcP homologs and the highest scoring GlcP Blast hits in symbiotic cyanobacteria. (S.I., sequence identity.).
| Strain | Annotation | Query cover | E-value | S.I. | Acces. No. |
|---|---|---|---|---|---|
| Top cyanobacterial Blast hits | |||||
| Nostoc punctiforme PCC 73102 | sugar transporter | 100% | 0 | 100% | YP_001868585.1 |
| Fischerella muscicola | major facilitator transporter | 100% | 0 | 88 | WP_016862018.1 |
| Crinalium epipsammum PCC 9333 | sugar transporter | 99% | 0 | 84% | YP_007144676.1 |
| Synechocystis sp. PCC 7509 | MFS transporter, sugar porter family | 99% | 0 | 83% | WP_009632434.1 |
| Nodosilinea nodulosa | major facilitator transporter | 99 | 0 | 80% | WP_017297719.1 |
| Cyanothece sp. PCC 7822 | sugar transporter | 99% | 0 | 74% | YP_003899859.1 |
| Chlorogloeopsis | major facilitator transporter | 100 | 0 | 74% | WP_016876260.1 |
| Oscillatoria acuminata PCC 6304 | sugar family MFS transporter | 98% | 0 | 74% | YP_007088081.1 |
| Microcoleus sp. PCC 7113 | sugar family MFS transporter | 94% | 0 | 76% | YP_007119491.1 |
| Lyngbya sp. PCC 8106 | major facilitator transporter | 98% | 0 | 72% | WP_009784998.1 |
| Synechocystis sp. PCC 6803 | unnamed protein product | 99% | 0 | 71% | CAA34119.1 |
| Gloeocapsa sp. PCC 73106 | MFS transporter, sugar porter family | 98% | 0 | 69% | WP_006529968.1 |
| Moorea producens | major facilitator transporter | 98% | 0 | 64% | WP_008188284.1 |
| Prochlorococcus marinus str. MIT 9303 | hypothetical protein | 99% | 3E-172 | 55% | YP_001017959.1 |
| Synechococcus sp. RS9917 | major facilitator transporter | 100% | 1E-160 | 55% | WP_007100383.1 |
| Synechococcus sp. CC9311 | major facilitator transporter | 97% | 1E-145 | 56% | WP_011618711.1 |
| Synechococcus sp. WH 8016 | sugar transporter | 41% | 2E-76 | 61% | WP_006854589.1 |
| Synechococcus sp. WH 8016 | general substrate transporter | 52% | 1E-64 | 53% | WP_006854590.1 |
| Symbiotic cyanobacterial Blast hits | |||||
|---|---|---|---|---|---|
| 'Nostocazollae' 0708 | EmrB/QacA transporter | 25% | 7e-07 | 30% | YP_003721762.1 |
| 'Nostocazollae' 0708 | major facilitator superfamily protein | 32% | 0.022 | 24% | YP_003721661.1 |
| cyanobacterium UCYN-A | glycosyl transferase | 6% | 0.13 | 42% | YP_003421499.1 |
| cyanobacterium UCYN-A | arabinose efflux permease family protein | 21% | 0.34 | 26% | YP_003421471.1 |
Except for the 12 cyanobacterial sequences, most homologous sequences were retrieved from Deltaproteobacteria, Actinobacteria, and Gammaproteobacteria. These sequences also show considerable sequence similarity (50–68% identity), and thus GlcP appears to be a widespread and conserved type of transporter. We constructed a phylogenetic tree over the 100 sequences with highest sequence similarity to N. punctiforme GlcP. The top scoring cyanobacterial sequences all cluster together and may have been acquired from Deltaproteobacteria (Fig. 2). The strains containing these GlcP sequences do however not cluster together in the cyanobacterial 16S phylogenetic tree (compare for example with Shih et al.),15 but are instead found in several distantly related branches. The presence and distribution of GlcP in cyanobacteria thus likely reflect events of intra-phylum horizontal gene transfer and possibly gene loss, with an unknown contribution of each of these mechanisms. The analysis also indicates that 2 additional independent acquisitions of GlcP-type permeases may have happened in cyanobacteria, one taking a gammaproteobacterial protein into Moorea producens and another taking an actinobacterial protein into Synechococcus/Prochlorococcus.
Figure 2. Phylogenetic tree of the 100 top scoring sequences in GlcP Blast search against the nr database (NCBI). The sequences were aligned with Muscle software (http://www.drive5.com/muscle/) using default settings. The resulting alignments were used for constructing an approximately-maximum-likelihood phylogenetic tree with the Fasttree software using default settings (http://www.microbesonline.org/fasttree/). The tree was rooted with the E. coli LacY transporter, an MFS transporter of a different type (family) than GlcP. Cyanobacterial strains are shown in black text while clusters of other bacterial phyla are collapsed and shown in color. *In addition to 49 actinobacterial sequences this cluster also contains 6 gammaproteobacterial and one firmicutes sequence. **In addition to 17 gammaproteobacterial sequences this cluster also contains one verrucomicrobial sequence.
The GlcP permease can have a nutritional role, facilitating heterotrophic or mixotrophic growth, in most cyanobacteria in which it is present, and additionally appears to have been recruited to accomplish symbiotic functions in N. punctiforme. These adaptations seem to have included changes in regulatory patterns, since the regulation of GlcP appears to differ between N. punctiforme and the non-symbiotic cyanobacteria.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank JC Meeks for sharing unpublished data. Research was supported by grant no. BFU2011–22762 from Plan Nacional de Investigación, Spain, co-financed by FEDER, and by The Swedish Research Council Formas.
Glossary
Abbreviations:
- MCP
methyl-accepting chemotaxis protein
References
- 1.Meeks JC, Campbell EL, Summers ML, Wong FC. Cellular differentiation in the cyanobacterium Nostoc punctiforme. Arch Microbiol. 2002;178:395–403. doi: 10.1007/s00203-002-0476-5. [DOI] [PubMed] [Google Scholar]
- 2.Bergman B, Ran L, Adams DG. Cyanobacterial-plant symbiosis: signal and development. In: Herrero A, Flores E, eds. The Cyanobacteria: Molecular Biology, Genomics and Evolution. Caiser Academic Press: Norfolk, UK, 2008:447-473 [Google Scholar]
- 3.Ekman M, Picossi S, Campbell EL, Meeks JC, Flores E. A Nostoc punctiforme sugar transporter necessary to establish a cyanobacterium-plant symbiosis. Plant Physiol. 2013;161:1984–92. doi: 10.1104/pp.112.213116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ungerer JL, Pratte BS, Thiel T. Regulation of fructose transport and its effect on fructose toxicity in Anabaena spp. J Bacteriol. 2008;190:8115–25. doi: 10.1128/JB.00886-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Schmetterer G, Flores E. Uptake of fructose by the cyanobacterium Nostoc sp. ATCC 29150. Biochim Biophys Acta. 1988;942:33–7. doi: 10.1016/0005-2736(88)90271-4. [DOI] [Google Scholar]
- 6.Meeks JC. Molecular mechanisms in the nitrogen-fixing Nostoc-bryophyte symbiosis. Prog Mol Subcell Biol. 2006;41:165–96. doi: 10.1007/3-540-28221-1_9. [DOI] [PubMed] [Google Scholar]
- 7.Nilsson M, Rasmussen U, Bergman B. Cyanobacterial chemotaxis to extracts of host and nonhost plants. FEMS Microbiol Ecol. 2006;55:382–90. doi: 10.1111/j.1574-6941.2005.00043.x. [DOI] [PubMed] [Google Scholar]
- 8.Khamar HJ, Breathwaite EK, Prasse CE, Fraley ER, Secor CR, Chibane FL, Elhai J, Chiu WL. Multiple roles of soluble sugars in the establishment of Gunnera-Nostoc endosymbiosis. Plant Physiol. 2010;154:1381–9. doi: 10.1104/pp.110.162529. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Ditty JL, Harwood CS. Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions. J Bacteriol. 1999;181:5068–74. doi: 10.1128/jb.181.16.5068-5074.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Hawkins AC, Harwood CS. Chemotaxis of Ralstonia eutropha JMP134(pJP4) to the herbicide 2,4-dichlorophenoxyacetate. Appl Environ Microbiol. 2002;68:968–72. doi: 10.1128/AEM.68.2.968-972.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ran L, Larsson J, Vigil-Stenman T, Nylander JA, Ininbergs K, Zheng WW, Lapidus A, Lowry S, Haselkorn R, Bergman B. Genome erosion in a nitrogen-fixing vertically transmitted endosymbiotic multicellular cyanobacterium. PLoS One. 2010;5:e11486. doi: 10.1371/journal.pone.0011486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Hilton JA, Foster RA, Tripp HJ, Carter BJ, Zehr JP, Villareal TA. Genomic deletions disrupt nitrogen metabolism pathways of a cyanobacterial diatom symbiont. Nat Commun. 2013;4:1767. doi: 10.1038/ncomms2748. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Thompson AW, Foster RA, Krupke A, Carter BJ, Musat N, Vaulot D, Kuypers MM, Zehr JP. Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga. Science. 2012;337:1546–50. doi: 10.1126/science.1222700. [DOI] [PubMed] [Google Scholar]
- 14.Muñoz-Marín MdelC, Luque I, Zubkov MV, Hill PG, Diez J, García-Fernández JM. Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean. Proc Natl Acad Sci U S A. 2013;110:8597–602. doi: 10.1073/pnas.1221775110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, Calteau A, Cai F, Tandeau de Marsac N, Rippka R, et al. Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci U S A. 2013;110:1053–8. doi: 10.1073/pnas.1217107110. [DOI] [PMC free article] [PubMed] [Google Scholar]