A Nostoc punctiforme Sugar Transporter Necessary to Establish a Cyanobacterium-Plant Symbiosis1[C][W] Martin Ekman 2,3, Silvia Picossi 2, Elsie L. Campbell, John C. Meeks, and Enrique Flores* Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, E–41092 Seville, Spain (M.E., S.P., E.F.); and Department of Microbiology, University of California, Davis, California 95616 (E.L.C., J.C.M.)
In cyanobacteria-plant symbioses, the symbiotic nitrogen-fixing cyanobacterium has low photosynthetic activity and is supplemented by sugars provided by the plant partner. Which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis, however, is unknown. Mutants of the symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme were constructed bearing a neomycin resistance gene cassette replacing genes in a putative sugar transport gene cluster. Results of transport activity assays using 14C-labeled fructose and glucose and tests of heterotrophic growth with these sugars enabled the identification of an ATP-binding cassette-type transporter for fructose (Frt), a major facilitator permease for glucose (GlcP), and a porin needed for the optimal uptake of both fructose and glucose. Analysis of green fluorescent protein fluorescence in strains of N. punctiforme bearing frt::gfp fusions showed high expression in vegetative cells and akinetes, variable expression in hormogonia, and no expression in heterocysts. The symbiotic efficiency of N. punctiforme sugar transport mutants was investigated by testing their ability to infect a nonvascular plant partner, the hornwort Anthoceros punctatus. Strains that were specifically unable to transport glucose did not infect the plant. These results imply a role for GlcP in establishing symbiosis under the conditions used in this work.
Living together in association, symbiosis, is an important mechanism of organization in the biological world. Symbiosis has been a key factor in biological evolution and is a driving force in many ecosystems in the biosphere. In associations involving a microbial symbiont, the symbiosis is frequently established de novo by uptake of the symbiont by the partner from the environment, whereas in others, the symbiont is transmitted vertically between partner generations (Bright and Bulgheresi, 2010). The cyanobacteria, prokaryotes that perform oxygenic photosynthesis, are prone to establish symbiotic associations, such as those found with fungi (as in lichens), diatoms, or plants. In cyanobacteriaterrestrial plant symbioses, the symbiotic cyanobacterium is invariably a nitrogen (N2)-fixing species that provides the plant with fixed nitrogen (Bergman et al., 1
This work was supported by the Dirección General de Investigación y Gestión del Plan Nacional de Investigación, Desarrollo e Innovación, Spain, and the European Regional Development Fund (grant nos. BFU2010–17980 and BFU2011–22762), by The Swedish Research Council Formas, and by the U.S. National Science Foundation (grant no. IOS 0822008). 2 These authors contributed equally to the article. 3 Present address: Department of Ecology, Environment, and Plant Sciences, Stockholm University, SE–106 91 Stockholm, Sweden. * Corresponding author; e-mail efl
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Enrique Flores (efl
[email protected]). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.112.213116 1984
2008). The cyanobacterium in the symbiosis has a low photosynthetic activity and in turn is supplemented by the plant partner with sugars (Adams and Duggan, 2008; Meeks, 2009). Although Fru and Glc have been suggested to be the sugars supplied by some plant partners (Wouters et al., 2000; Khamar et al., 2010), exactly which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis is unknown. Species of the genus Nostoc are frequently found in symbiosis (Bergman et al., 2008). Cyanobacteria of this genus grow forming chains of cells (filaments or trichoma) in which, under conditions of combined nitrogen deprivation, some cells differentiate into N2-fixing heterocysts (Flores and Herrero, 2010). Nostoc spp. vegetative cells can also differentiate into akinetes, a kind of spore, or hormogonia, which are short filaments made of smaller cells that function in dispersal by a gliding motility (Meeks et al., 2002). Hormogonia are the infective units in symbiosis through which Nostoc spp. establishes de novo associations (Meeks and Elhai, 2002; Adams and Duggan, 2008). Hormogonia can differentiate in response to a variety of environmental signals, and consistent with their role in establishing symbiosis, they can also differentiate in response to a hormogonium-inducing factor (HIF) produced by some plant partners (Campbell and Meeks, 1989). Anthoceros punctatus is a nonvascular plant of the division Anthocerotophyta (hornworts) whose gametophytes support the growth of microcolonies of Nostoc spp., formed after infection of the plant tissue by hormogonia (Meeks and Elhai, 2002). The symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme (strain ATCC 29133 or PCC 73102),
Plant PhysiologyÒ, April 2013, Vol. 161, pp. 1984–1992, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved.
Sugar Transporter in a Nostoc punctiforme-Plant Symbiosis
originally isolated from a root section of the cycad Macrozamia spp. (Rippka et al., 1979), can establish symbiosis with A. punctatus (Enderlin and Meeks, 1983). The complete genomic sequence of N. punctiforme has been determined and is available (http://img.jgi.doe. gov/; Meeks et al., 2001). This organism can receive DNA from Escherichia coli by conjugation (Flores and Wolk, 1985) and is amenable to genetic analysis (Cohen et al., 1994). Therefore, it is an appropriate model organism in which to study aspects of symbiotic association. We are interested in understanding the molecular basis of sugar nutrition in the symbiont of an A. punctatus association. Many strains of the genus Nostoc are facultative heterotrophs (Rippka et al., 1979), and the sugars most frequently assimilated by Nostoc spp. and other cyanobacteria are Fru, Glc, and Suc (Rippka et al., 1979). Fru or Glc transport activity has been reported for some Nostoc spp. strains (Beauclerk and Smith, 1978; Schmetterer and Flores, 1988), including Nostoc sp. filaments isolated from a symbiotic association (Black et al., 2002). These sugars have also been shown to support nitrogenase activity in a Nostoc sp. strain UCD 7801-A. punctatus association (Steinberg and Meeks, 1991). Only two sugar transporters from other cyanobacteria have been identified at the molecular level. One is the major facilitator superfamily Glc permease of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803), named GlcP (Zhang et al., 1989) or Gtr (Schmetterer, 1990), which might also mediate Fru uptake (Flores and Schmetterer, 1986). The second is an ATP-binding cassette (ABC)-type Fru transporter from the heterocyst-forming cyanobacterium Anabaena variabilis (Ungerer et al., 2008). This transporter is the product of the genes in a putative operon, frtABC, encoding a periplasmic binding protein (FrtA), an ATPase subunit (FrtB), and an integral membrane protein (FrtC). Adjacent to frtABC, on the opposite DNA strand, is frtR, encoding a LacI-like transcription factor that regulates the expression of the operon (Ungerer et al., 2008). Genes encoding homologs to the Frt transporter proteins and to GlcP are present in a gene cluster in the genome of N. punctiforme, and a gene encoding a putative sugar-specific porin is adjacent to these genes. Interestingly, also adjacent to this cluster of genes is a cluster of genes (called hrm) whose products are involved in the suppression of hormogonium differentiation (Cohen and Meeks, 1997; Meeks, 2006). One of these genes encodes HrmR, which is homologous to A. variabilis FrtR. These observations imply a possible relation of the sugar transport genes with hormogonium differentiation and/or symbiosis. In this work, we have created N. punctiforme mutants with altered expression of genes within this sugar transport-related cluster and analyzed their ability to establish symbiosis with A. punctatus. RESULTS Sugar Transporters
In the genome of N. punctiforme, a cluster of genes is present encoding homologs to the A. variabilis ABCPlant Physiol. Vol. 161, 2013
type Fru transporter (Frt), the Synechocystis 6803 Glc permease (GlcP), and a putative carbohydrate porin, OprB. We name these genes in N. punctiforme according to the homology of their encoded products. The genes encoding the ABC-type transporter complex are two frtA homologs (frtA1 and frtA2), one frtB, and one frtC (Fig. 1). Immediately downstream of frtC is glcP, and about 1 kb downstream of the latter is oprB. Mutants of N. punctiforme were constructed that bore a neomycin resistance gene cassette (C.K3) replacing most of the four frt genes, glcP, or oprB, as shown in Figure 1. A mutant in which a chromosomal fragment containing both glcP and oprB was replaced by C.K3 was also obtained. The mutants were generated after transfer of the corresponding constructs by conjugation from E. coli to the N. punctiforme wild-type strain (ATCC 29133) or to a spontaneous derivative (ATCC 29133-S, also identified as strain UCD 153; Campbell et al., 2007) that grows more homogeneously and rapidly in liquid medium than the wild type but whose hormogonia are less active in gliding. In the C. K3 gene cassette, the npt gene encoding neomycin phosphotransferase is transcribed from the strong PpsbA promoter from the chloroplast of Amaranthus hybridus (Elhai and Wolk, 1988). Because this gene cassette bears no transcription terminators and, therefore, may produce polar effects, we prepared the different constructs (except the one removing glcP and oprB together) with the gene cassette inserted in both orientations (Fig. 1). All the mutant strains were homozygous, containing only mutant chromosomes, as shown by PCR analysis (Supplemental Fig. 1). Filaments of N. punctiforme strains ATCC 29133 and ATCC 29133-S (UCD 153) grown in BG110 + NH4+ medium showed uptake of [14C]Fru and [14C]Glc linearly for at least 1 h, with strain UCD 153 exhibiting somewhat higher activities than the wild type (Supplemental Fig. S2). To test the possible role of the identified genes in sugar transport, the uptake of these labeled substrates was determined in the mutants and compared with their respective parental strains. Strains in which the frt genes were removed (CSME1A, CSME1B, and CSME-S1A) showed very low uptake of Fru (Table I), consistent with the notion that Frt is a Fru transporter. Fru uptake was also impaired (42%–76% of the activity of the corresponding parental strain) in strain CSME11, in which both GlcP and OprB were removed, and in strains CSME-S12A and CSME-S12B, in which only OprB was removed. These results imply that OprB is at least needed for optimal Fru uptake, consistent with the idea that this protein is a sugar porin. Strains CSME-S13A and CSME-S13B, in which only GlcP was removed, showed a low and an intermediate rate of Fru uptake, respectively. Whereas the results with strain CSME-S13B may imply that GlcP also mediates Fru transport, the results with CSME-S13A most likely indicate a further effect of C.K3, which in the latter strain was inserted in the opposite orientation to the genes (see below). Strains CSME-S13A and CSME-S13B showed very low levels of Glc uptake (Table I), consistent with the 1985
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Figure 1. Structure of the sugar transport genomic region in N. punctiforme and mutants generated in this work. ORF numbers (Npun_) and proposed gene names are indicated for each gene. Two putative pseudogenes, Npun_R5321 and Npun_R5322 (not shown here), can be found between Npun_R5320 and Npun_R5323. The site of insertion and the orientation of the C.K3 gene cassette (gray arrows) are shown for the different constructs, and the names of the N. punctiforme strains bearing those constructs are indicated. Note that in each case, the gene cassette replaced a DNA fragment in the chromosome. In the bottom right corner, the DNA fragments used to promote gfp expression in N. punctiforme strains carrying the corresponding constructs in plasmid pRL25C are depicted. The sizes of the genes and intergenic regions are shown.
notion that GlcP is a Glc transporter, and strains CSME-S12A and CSME-S12B were impaired in Glc uptake to a similar extent as in Fru uptake, corroborating that OprB may function as a sugar porin. Strain CSME11, which lacks both GlcP and OprB, consistently showed a very low rate of Glc uptake. On the other hand, the effect on Glc uptake of replacing the frt genes by C.K3 was strongly influenced by the orientation of the gene cassette. When C.K3 was oriented opposite to that of the genes in the gene cluster (as in strains CSME1A and CSME-S1A), the rate of Glc uptake was very low. When C.K3 was in the same orientation as the genes in the gene cluster (as in strain CSME1B), an activity 6-fold higher than that of the parental strain was observed. We hypothesized that the latter effect is a
consequence of transcription from the gene cassette increasing expression of the downstream genes. Northernblot analysis with probes of either glcP or oprB showed an exceptionally high expression of these genes in strain CSME1B (Supplemental Fig. S3), which can account for the observed increase in Glc uptake (Table I). Insertion of the gene cassette into glcP, oriented opposite to the frt genes (as in the CSME-S13A strain), resulted in a low rate of Fru uptake; high expression from C.K3 could result in negative effects on transcription, mRNA stability, or translation. The results obtained indicate that the putative ABCtype Frt transporter does mediate Fru uptake, the identified GlcP is a Glc permease, and the OprB-like porin is needed for the optimal uptake of both Fru and
Table I. Fru and Glc uptake and symbiotic performance in N. punctiforme parental strains and mutants Fru Uptakea
Strain
nmol mg21 chlorophyll min21
ATCC 29133 CSME1A CSME1B CSME11 UCD 153 CSME-S1A CSME-S12A CSME-S12B CSME-S13A CSME-S13B
11.6 0.3 0.9 4.9 38.0 0.4 29.0 24.1 10.1 17.8
6 6 6 6 6 6 6 6 6 6
7.1 (3) 0.1 (3) 0.1 (3) 3.3 (3) 18.8 (8) 0.1 (3) 7.5 (6) 10.1 (5) 8.5 (6) 2.2 (4)
Glc Uptakea %
100 3 8 42 100 1 76 63 27 47
nmol mg21 chlorophyll min21
21.2 1.3 125.6 1.2 44.0 1.7 22.3 32.1 0.6 0.9
6 10.2 (4) 6 0.3 (4) 6 47.6 (4) 6 0.4 (4) 6 10.4 (8) 6 0.6 (4) 6 5.4 (5) 6 7.7 (5) 6 0.4 (5) 6 0.02 (3)
Symbiotic Phenotypeb %
100 6 592 6 100 4 51 73 1 2
+ 2 + 2 + 2 + + 2 2
Transport assays were carried out with 100 mM substrate for 40 to 60 min as described in “Materials and Methods.” Each number is the mean and b of the results from the number of experiments performed with independent cultures indicated in parentheses. The symbiotic phenotype was scored as positive if the presence of symbiotic colonies in plant tissue was observed after 2 weeks of coculture. For about 5 g of fresh plant tissue, about 400 to 600 colonies were observed for ATCC 29133 and its positive derivatives and 60 to 100 colonies for UCD 153 and its positive derivatives (three flasks were tested for each cyanobacterial strain). The phenotype was scored as negative if no colonies were observed. Symbiotic performance was confirmed in each case by the presence of dark green plant tissue after 4 weeks of coculture. a
SD
1986
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Glc. Results of growth analysis with sugar supplementation were consistent with the transport data. As shown in Figure 2, N. punctiforme grew heterotrophically better with Fru than with Glc, which corroborates the results of Summers et al. (1995), but growth did not take place in the CSME1A strain that is hampered in the transport of both substrates. Overexpression of glcP and oprB in strain CSME1B allowed an increased growth performance, and likely also hastened hormogonia formation (as deduced from the spreadable aspect of the spot; Fig. 2), of this strain specifically on Glc. Finally, strain CSME11 could not grow with Glc but showed some growth with Fru, consistent with its lack of activity of Glc transport and its partial activity of Fru transport. Heterotrophic growth that was more robust with Fru than with Glc was also observed with strain UCD 153 (data not shown).
heterocysts, GFP fluorescence was extremely low or null (Fig. 3, E and F). High GFP expression in vegetative cells and lack of expression in heterocysts was corroborated in strain CSME-S18 (data not shown), which carries only the region upstream from frtA1, indicating that the regulated expression of the frt genes in heterocysts versus vegetative cells does not require sequences upstream from hrmE. In these analyses, the cultures did not contain any added sugar, indicating that expression from the frtA1 promoter does not require induction by a sugar. There are two HrmR-binding sites in and near the promoter of hrmE but not upstream of frtA1 (Meeks, 2006); therefore, HrmR, the homolog of FrtR, does not appear to be involved in the transcriptional regulation of the frt genes. Symbiotic Phenotype
Cell Specificity of Expression
To investigate the cell type in which the sugar transport genes are expressed in the filaments of N. punctiforme, a fusion of the gfp-mut2 gene encoding a GFP to the seventh codon of frtA1 was prepared in two constructs. One DNA fragment covering the intergenic region between hrmE and frtA1 and another fragment extending backward to the region upstream of hrmE were used to prepare strains CSME-S18 and CSME-S19, respectively (Fig. 1). The corresponding constructs were cloned in pRL25C, a shuttle vector that can replicate in both E. coli and some filamentous, heterocyst-forming cyanobacteria (Wolk et al., 1988), and transferred to N. punctiforme strain UCD 153 by conjugation from E. coli. The construct carrying the region upstream from hrmE through frtA1 (in strain CSME-S19) showed strong GFP fluorescence in vegetative cells of ammonium-grown filaments (Fig. 3, A–C) or of filaments incubated in the absence of a source of combined nitrogen (Fig. 3, E and F), although some variability in fluorescence was observed between cells. GFP fluorescence was very high in akinetes (Fig. 3, A, B, and G). Regarding hormogonia, filaments exhibiting either high (Fig. 3C) or low (Fig. 3D) GFP fluorescence were observed, but we did not attempt a systematic study of these differences. In
Figure 2. Heterotrophic growth with sugars of N. punctiforme strain ATCC 29133 (WT) and some sugar transport mutants (CSME strain numbers). Filament samples were spotted in BG110 + NH4+ solid medium supplemented with 5 mM Fru (Frc) or 5 mM Glc and incubated in the dark for 2 weeks. Plant Physiol. Vol. 161, 2013
Hormogonia are the infection units of N. punctiforme in the establishment of a plant symbiosis, and in the symbiosis the cyanobacterium provides the plant with fixed nitrogen. All the sugar transport mutants isolated in this study produced hormogonia and were able to grow diazotrophically in the light with CO2 as the carbon source (data not shown). The symbiotic phenotype of the mutants was investigated by adding each mutant to an A. punctatus culture that had been growing on nitrogen-free medium supplemented with 28 mM Glc for 7 to 10 d. Glc was added for optimal growth of A. punctatus under the light intensity of 31.5 mmol m22 s21. The infection frequency was scored after 2 weeks of coculture of the cyanobacteria with the plant by counting cyanobacterial colonies established in the plant tissue, and symbiotic performance was deduced from a dark green color of the plant tissue showing evidence of nitrogen supply (Fig. 4). The results obtained with the different mutants are summarized in Table I. N. punctiforme mutants that showed a very low activity of Glc transport or a low activity of both Glc and Fru transport did not infect A. punctatus. Thus, all the glcP mutants and the frt mutants in which C.K3 was in the opposite orientation to the sugar transport genes did not infect the plant. In contrast, strain CSME1B, which showed a very low Fru transport, while overexpressing glcP and oprB, showed infection. The mutants in which only OprB was removed were also infective. Symbiotic performance was further investigated in a 4-week test for those mutants that had been observed to infect the plant. The GlcPdefective strain CSME13A, which showed the lowest Glc transport activity, was used as a negative control (Fig. 4). All mutants that infected were effective in symbiotic function by providing the plant with nitrogen. These results indicate a need for the GlcP permease for establishing the symbiosis. DISCUSSION
N. punctiforme strain ATCC 29133 was originally isolated as a symbiont from a cycad. It can grow in the 1987
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Figure 3. Expression of GFP in N. punctiforme strain CSME-S19 bearing an frtA1-gfp-mut2 translational fusion. Filaments of CSME-S19 were grown in BG110 + NH4+ medium (A–C) and, after washing with BG110 medium (lacking any source of combined nitrogen), incubated in BG110 medium for the indicated times (D–G). Filaments were visualized by confocal microscopy with identical settings for all samples. Bright-field (left panels), red cyanobacterial autofluorescence (middle panels), and green GFP fluorescence (right panels) images are shown. No green fluorescence was observed with the parental strain UCD 153 (data not shown). Filaments in A to C are composed mostly of vegetative cells. Brackets in A, B, and G indicate an akinete or akinete rows. The boxed filament in C is a hormogonium, and the filament in D is in part differentiating into a hormogonium (note enclosure). Arrowheads in E and F point to heterocysts (which lack autofluorescence) that are found next to vegetative cells. Bars = 10 mm (E–G), 20 mm (A), and 40 mm (B–D). 1988
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Figure 4. Coculture of A. punctatus with examples of N. punctiforme sugar transport mutants. The top image shows flasks containing about 5 g of fresh plant tissue in nitrogen-free growth medium inoculated with the indicated cyanobacterial strains. The bottom images are micrographs of tissue from the flasks above, in which some cyanobacterial colonies are highlighted by circles. Photographs were taken after 4 weeks of coculture.
free-living state autotrophically, photoheterotrophically, or dark heterotrophically using Glc, Fru, Rib, or, weakly, Suc (Rippka et al., 1979; Summers et al., 1995). We have shown that Glc uptake is mediated by the GlcP major facilitator superfamily permease and Fru uptake by the FrtA1-FrtA2-FrtB-FrtC ABC-type transporter. The homologous Glc permease from Synechocystis 6803 might also mediate Fru uptake (Flores and Schmetterer, 1986; Zhang et al., 1989). Replacing the glcP gene with the C.K3 cassette inserted in the same orientation as the frt genes (in strain CSME-S13B) results in moderately impaired Fru uptake. This could imply that GlcP may also aid Fru uptake in N. punctiforme. However, if the frt genes and glcP form an operon, the possibility cannot be ruled out that replacing glcP by the gene cassette affects the stability of the part of the transcript containing the frt genes, with the effect of diminishing translation and production of the Frt transporter complex. The very low activity of Glc uptake in strains CSME1A and CSMES1A, which interrupt the frt genes by antiparallel insertion of the C.K3 cassette, may result from poor expression of the glcP gene in these mutants as a consequence of antisense interference with a transcript initiated in a promoter 59 of frtA1. If so, the frt and glcP genes could be transcribed as an operon. Regarding the gene located downstream from the putative frt-glcP operon, because its inactivation impairs the uptake of both Fru and Glc independently of the sense in which the C.K3 cassette is inserted, we infer that the encoded protein facilitates the uptake of both sugars. This is consistent with the identification based on homology of this protein as OprB, a Plant Physiol. Vol. 161, 2013
carbohydrate-selective porin originally characterized in Pseudomonas putida (Saravolac et al., 1991). As investigated with a translational fusion of the GFP to the N-terminal part of FrtA1 encoded in a replicating plasmid, the frt-glcP genes appear to be expressed at high levels in vegetative cells and akinetes and at variable levels in hormogonia, whereas expression in heterocysts appears to be null. Expression in vegetative cells is consistent with the detection of sugar uptake activity in the ammonium-grown cultures used in this work. However, some variability in expression was observed along the filaments. Differences in plasmid copy number between cells could affect the expression levels detected, and this should be considered also when comparing different cell types (Argueta et al., 2004). In particular, akinetes of some cyanobacteria have been shown to accumulate considerable amounts of genetic material (Adams and Duggan, 1999; Sukenik et al., 2012), which could increase the detection of a gene expressed in these differentiated cells. On the other hand, hormogonial cells (formed by cell division without growth) have lower amounts of genetic material than vegetative cells (Herdman and Rippka, 1988), which could affect the level of detection of an expressed gene or operon. According to microarray analysis of hormogonia induced by nitrogen stress or by the presence of HIF, the open reading frames (ORFs) encoding the sugar transporters are under complex regulation (Campbell et al., 2008). Whereas nitrogen stress provoked a slow induction over a period of 24 h, HIF elicited an early (less than 1 h) strong induction followed by a slow decline in transcription of those ORFs over a period of 24 h. The hormogonia showing different levels of fluorescence from the FrtA1-GFP fusion could be at different stages of development. The symbiotic phenotype that we have analyzed involves colonization by N. punctiforme hormogonia of
Figure 5. Schematic of the Nostoc spp.-plant symbiotic interaction. The cyanobacterium forms many more heterocysts (double-circled cells) in symbiosis than in the free-living state. In the A. punctatus symbiosis, nitrogen (N) fixed in the heterocysts is transferred in the form of ammonia to the plant by an unknown molecular mechanism (Meeks et al., 1985). The plant provides the cyanobacterium with sugar. As shown in this work, the GlcP permease expressed in the vegetative cells of the cyanobacterium is essential for the symbiosis, implying a role of Glc and of any other possible substrate of the permease in the symbiosis. C, Carbon. [See online article for color version of this figure.] 1989
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the A. punctatus gametophyte tissue and growth of the symbiont to produce visible cyanobacterial colonies. In the cluster of genes analyzed in this work (ORFs Npun_R5327 to Npun_R5320), only glcP is essential for the establishment of the symbiosis under our experimental conditions. The GlcP permease may be required for colonization and/or for nutrition of the symbiont in the plant. The glcP mutant represents the only reported instance in which a mutant of any heterocyst-forming cyanobacterium able to fix N2 in the free-living state and also able to form hormogonia is unable to establish a nitrogen-fixing symbiotic association. Previously tested strains of N. punctiforme unable to form functional associations were mutants that cannot fix N2 in air in the free-living state (Meeks, 2006). These include mutants of the ntcA, hetR, and hetF genes that encode positive regulators of heterocyst differentiation. The devR mutant, impaired in the production of the heterocyst envelope polysaccharide layer, cannot fix N2 under oxic conditions in the free-living state, but the mutant infects and fixes N2 in symbiosis (Campbell et al., 1996). Mutants such as those defective in hetR or hetF are able to differentiate hormogonia and colonize the plant tissue, but the resulting colonies do not fix N2 (Wong and Meeks, 2002). Only the ntcA mutant resembles the glcP mutant in not establishing appreciable colonies in the plant tissue (Wong and Meeks, 2002). The absence of GlcP, therefore, appears to affect cellular processes during the establishment of symbiosis under our assay conditions, thus preceding a nitrogen fixation activity that is dependent on externally supplied sugars. The ORFs in the sugar-uptake gene cluster are induced by the hormogonium-repressing factor (Meeks, 2006), which also appears to be present in the plant exudate containing HIF (Campbell et al., 2008). This would imply an induction of the sugar-uptake capability when hormogonia differentiation is suppressed in planta. On the other hand, overexpression of glcP, as in strain CSME1B, results in a very high activity of Glc uptake (Table I) and increased heterotrophic growth with Glc of free-living N. punctiforme (Fig. 2). This observation raises the possibility that an increased expression of this gene (or perhaps of the whole operon) in planta facilitates the growth of N. punctiforme in symbiosis. An important role of the GlcP permease and its associated Glc uptake by the symbiont is consistent with the results of a previous study in which a significant fraction of Glc taken up by infected tissue of a Gunnera tinctoriaNostoc sp. symbiosis was observed to correspond to assimilation by the cyanobacterium (Black et al., 2002). In another study, nitrogen-deprived Gunnera manicata plants were found to accumulate high levels of Glc and Fru. However, N. punctiforme colonization drastically reduced the levels of these sugars at the colonization sites in the plant (Khamar et al., 2010). Our results do not rule out the possibility that Fru or any other sugar that can be assimilated by N. punctiforme, such as Suc or Rib, has a role in nutrition of the 1990
cyanobacterial symbiont. Decreased Fru uptake, as in strain CSME1B, did not by itself affect symbiotic performance, but the high activity of Glc uptake taking place in this strain might compensate for the missing Fru uptake. In symbiosis, a high percentage of the cells in a filament are heterocysts (Meeks and Elhai, 2002). We have observed by GFP-translational fusions that in free-living N. punctiforme, the frt-glcP-encoded proteins are expressed in vegetative cells but not in heterocysts. If this cellular localization pattern were maintained in planta, nutrition of the filament in the symbiosis would involve uptake of the sugar specifically by the vegetative cells (Fig. 5). This is consistent with the results of studies carried out on the Gunnera-Nostoc spp. symbiosis (Black et al., 2002). Uptake specifically by vegetative cells would imply that the mechanisms that may operate to transfer sugars from the vegetative cells to heterocysts in the free-living filament are also operative in the symbiotic filament. If so, a contiguous heterocyst distribution in the filaments would be much less efficient in reductant supply for nitrogen fixation than one in which a single heterocyst is bound by two or more vegetative cells. The former pattern is seen in older infected plant tissue or older portions of symbiotic Nostoc spp. colonies, while the latter is seen in the regions of highest nitrogen fixation activity (Hill, 1975). Our results show that sugar transport via GlcP is essential for establishing the symbiosis between a cyanobacterium and a plant, but sugars have been implicated also in other aspects of the symbiosis. Sugars have been suggested to have a role as chemoattractants for the cyanobacteria during the infection process (Rasmussen et al., 1996; Nilsson et al., 2006; Khamar et al., 2010). It has also been suggested that various sugars, including Glc and Fru, repress the formation of hormogonia after the symbiosis has been established (Khamar et al., 2010). If the high levels of such sugars present in symbiotic glands of Gunnera spp. act in this way, then one might expect to find a regulatory connection between the hrm genes and the sugar transporters. Indeed, the 59 region of frtA1 and hrmI share a conserved sequence (TGAAAACTGTAGTTT) that in hrmI is located between the 210 and 235 RNA polymerase s-subunit recognition sequences, where binding of a putative regulatory protein would inhibit transcription; such binding has not been experimentally determined (Meeks, 2006). Finally, because a high cellular carbon-to-nitrogen balance induces heterocyst differentiation (Flores and Herrero, 2010), it has been speculated that the sugars supplied by the plant partner may enhance heterocyst differentiation in symbiotic Nostoc spp. (Meeks, 2006). In summary, sugar uptake and metabolism may have a role not only in nutrition of the cyanobacterial symbiont but also in regulatory mechanisms important in establishing and maintaining the symbiosis. The results of this study provide a molecular basis for further investigations aimed at understanding these additional roles of sugars in plantcyanobacterial symbioses. Plant Physiol. Vol. 161, 2013
Sugar Transporter in a Nostoc punctiforme-Plant Symbiosis
MATERIALS AND METHODS
Transport Assays
Bacterial Strains and Growth Conditions
For 14C-labeled sugar-uptake assays, N. punctiforme parental and mutant strains were grown in BG110/NH4+/TES medium. The cultures were harvested at room temperature, washed twice with BG110/NH4+/TES medium, and resuspended in the same solution to give a cell density corresponding to 10 mg chlorophyll a mL21. The assay was started by adding 0.2 mL of a sugar solution containing either 1.1 mM Glc and 15 mM [14C]Glc (300 Ci mol21; Perkin-Elmer) or 1.1 mM Fru and 15 mM [14C]Fru (300 Ci mol21; Perkin-Elmer) to a 2-mL cell suspension. The cultures were incubated at 30°C at a light intensity of 85 mmol m22 s21, and 0.5-mL samples were filtered, using 0.45-mm pore size Millipore HA filters, at the indicated time points. After washing with BG110/NH4+/TES to remove excess labeled sugar, the filters were placed in a scintillation cocktail, and their radioactivity was measured. Nonspecific retention of radioactivity was determined by using boiled cell samples.
Nostoc punctiforme strain ATCC 29133, or its derivative ATCC 29133-S (also known as UCD 153; Campbell et al., 2007, 2008), was grown in BG110 medium (BG11 medium; Rippka et al., 1979; free of combined nitrogen) or BG110 medium supplemented with 2.5 mM NH4Cl and 5 mM TES-NaOH buffer (pH 7.5) at 30°C in the light (25 mmol m22 s21) in shaken (100 rpm) liquid cultures or on medium solidified with 1% Difco agar. For the mutants described below, neomycin was used in liquid medium at 10 mg mL21 and in solid medium at 25 mg mL21. For heterotrophic growth assays, 20 mL of N. punctiforme strain cell suspensions was placed on plates containing either 5 mM Fru or 5 mM Glc, in addition to BG110/NH4+/TES/agar, and incubated in darkness. Escherichia coli strain DH5a, used for plasmid constructions, and strains HB101 and ED8654, used for conjugations with N. punctiforme strains, were grown in Luria-Bertani medium supplemented when appropriate with antibiotics at standard concentrations (Ausubel et al., 2012).
Construction of Cyanobacterial Mutants Genomic DNA was isolated from N. punctiforme as described previously (Cai and Wolk, 1990). For gene deletions in the sugar transporter gene cluster, 1-kb flanking regions of the DNA sequence to be deleted (Fig. 1) were amplified and fused by overlapping PCR, except for CSME11, where the flanking regions were fused by ligation after SmaI digestion (oligodeoxynucleotide primers are listed in Supplemental Table S1). This resulted in DNA fragments containing SacI and XhoI at each end, and SmaI in the middle (at the fusion site). XhoI/SacI-digested fragments were then cloned into XhoI/SacI-digested pRL271 (Black et al., 1993). The final constructs were obtained by inserting the gene cassette C.K3 encoding resistance to neomycin (Nmr) and resistance to kanamycin (Elhai and Wolk, 1988) at the SmaI site of the fragments (i.e. between the two flanking regions, either in direct [indicated by “B” at the end of the mutant strain names] or in opposite [indicated by “A” at the end of the strain names] orientation). To produce a GFP reporter strain for the localization of frtA1 gene expression, a fragment including the first seven codons of Npun_R5327 and 988 bp upstream of the same gene (including one-half of Npun_R5328) was amplified by PCR using primers NpR5327-GFP-ClaI and NpR5327-GFP-EcoRV (which contain a ClaI and an EcoRV restriction site, respectively) and N. punctiforme DNA as template. After digestion with ClaI and EcoRV, this fragment was cloned to ClaI/EcoRV-digested pCSEL21 (Olmedo-Verd et al., 2006), producing a translational fusion of the gfp-mut2 gene (Cormack et al., 1996) to the Npun_R5327 59 region. The resulting fusion was transferred as an EcoRI-ended fragment to EcoRI-digested pRL25C (Wolk et al., 1988), producing pCSME18 (Nmr). The same procedure as above was applied to produce a GFP reporter strain where gfp-mut2 was fused to a longer DNA fragment (2,144 bp upstream from Npun_R5327, including also the hrmE gene and its upstream region, extending 27 nucleotides into the divergent Npun_F5329 gene), except that primer NpR5328-GFP-ClaI instead of NpR5327-GFP-ClaI was used in the PCR amplification, resulting in plasmid pCSME19 (Nmr). In both cases, the 39 end of the fragment (i.e. the truncated reading frame of Npun_R5327) is fused to the gfp-mut2 in frame. Transfer of the final constructs (cargo plasmids) to N. punctiforme strains was performed by conjugation, based on previously described procedures (Elhai et al., 1997). Ten milliliters of E. coli strain HB101 carrying helper/methylation plasmid pRL623 and a cargo plasmid was harvested at exponential phase (optical density at 750 nm of approximately 1.0) and mixed with an equal amount of E. coli ED8654 carrying conjugative plasmid pRL443, giving a final volume of approximately 150 mL. After 1 h of mating, 100 mL of N. punctiforme culture, at a concentration of 175 mg chlorophyll a mL21, was added to the E. coli mixture, which was then plated on Millipore Immobilon NC filters placed on top of BG110/ NH4+/TES plus 5% Luria-Bertani plates. After a 24-h incubation at 30°C under low light (5 mmol m22 s21), the membranes were transferred to BG110/NH4+/TES plates and incubated at 25°C for 48 h in low light followed by 24 h in normal light (25 mmol m22 s21). Finally, the membranes were transferred to BG110/NH4+/ TES/Nm plates and incubated at 25°C, in normal light, until neomycin-resistant colonies appeared. For gene inactivations, double recombinants were selected from single recombinants by growth on plates containing 5% Suc, as described previously (Cai and Wolk, 1990). The genetic structure of the resulting N. punctiforme clones was confirmed by PCR with DNA from those clones and the primer pairs indicated in Supplemental Figure S1. Plant Physiol. Vol. 161, 2013
Microscopy N. punctiforme ATCC 29133 and strains CSME18 and CSME19 were incubated in liquid medium with or without a source of combined nitrogen. Samples from these cultures were analyzed in a Leica TCS SP5 confocal laserscanning microscope. The GFP emission, collected between 500 and 530 nm, was observed after excitation at 488 nm, whereas cyanobacterial autofluorescence was collected between 680 and 730 nm.
Plant Cultures and Reconstitution of Symbiosis Anthoceros punctatus gametophyte tissue was grown in basal medium (Enderlin and Meeks, 1983) buffered with 5 mM MES (pH 6.4) under a 16/8-h light/dark cycle (light intensity, 31.5 mmol m22 s21) and also supplemented with 0.5% (w/v) Glc for optimal growth. To reconstitute the symbiotic associations, A. punctatus tissue (about 5 g) was first incubated in 50 mL of Glcsupplemented basal medium minus ammonium nitrate for 7 to 10 d. N. punctiforme was cultured for reconstitution experiments under nitrogen-fixing conditions as before (Campbell and Meeks, 1989). Mutant and wild-type strains of N. punctiforme at a total chlorophyll a content of 15 to 20 mg were then combined with the nitrogen-starved A. punctatus tissue in the same conditioned medium. Symbiotic colonies in gametophyte tissue were counted with the aid of a dissecting microscope after coculturing for 2 weeks. The GenBank/EMBL accession number for the whole genomic sequence of N. punctiforme is CP001037.1. The accession numbers of the individual gene products reported in this article are: Npun_R5320 (ACC83641.1); Npun_R5323 (ACC83642.1); Npun_R5324 (ACC83643.1); Npun_R5325 (ACC83644.1); Npun_R5326 (ACC83645.1); Npun_R5327 (ACC83646.1); and Npun_R5328 (ACC83647.1).
Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Structure of the sugar transport genomic region in the mutants. Supplemental Figure S2. Sugar uptake time courses. Supplemental Figure S3. Overexpression of glcP and oprB in strain CSME1B. Supplemental Table S1. Oligodeoxynucleotide primers.
ACKNOWLEDGMENTS We thank Antonia Herrero for generous support and Jeff Elhai and Wan-Ling Chiu for critically reading the manuscript. Received December 19, 2012; accepted March 4, 2013; published March 5, 2013.
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