Plant Physiology Preview. Published on March 5, 2013, as DOI:10.1104/pp.112.213116
Running head:
Sugar transporter in a Nostoc-plant symbiosis
Corresponding author:
Enrique Flores
Instituto de Bioquímica Vegetal y Fotosíntesis,
CSIC and Universidad de Sevilla,
Américo Vespucio 49,
E-41092 Seville, Spain
Tel.: +34954489523
Fax: +34954460065
E-mail: [email protected]
Journal research area:
Plants Interacting with Other Organisms
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Copyright 2013 by the American Society of Plant Biologists
Nostoc sugar transporter necessary to establish a
cyanobacterium-plant symbiosis1
Martin Ekman‡,†, Silvia Picossi†, Elsie L. Campbell, John C. Meeks and
Enrique Flores*
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla,
Américo Vespucio 49, E-41092 Seville, Spain (M.E., S.P., E.F.), and Department of
Microbiology, University of California, Davis, CA 95616, USA (E.L.C., J.C.M.)
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1
Work in Seville was supported by grants BFU2010-17980 and BFU2011-22762
from Dirección General de Investigación y Gestión del Plan Nacional de I+D+I
(Spain), co-financed by FEDER. At UC Davis, the work was supported by U.S.
National Science Foundation grant IOS 0822008.
†
These authors contributed equally to this work.
‡
Present address: Department of Botany, Stockholm University, SE-106 91
Stockholm, Sweden
*Corresponding author. E-mail: [email protected]. 3
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ABSTRACT
In cyanobacterial-plant symbioses, the symbiotic N2-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 is, however, 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
14
C-labeled
fructose and glucose and tests of heterotrophic growth with these sugars enabled
the identification of an ABC-type transporter for fructose (Frt), a major facilitator
permease for glucose (GlcP), and a porin needed for optimal uptake of both
fructose and glucose (OprB). Analysis of GFP 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 non-vascular 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 the glucose permease, GlcP, in establishing
the symbiosis under the conditions used in this work.
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INTRODUCTION
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 cyanobacterial-terrestrial plant symbioses, the symbiotic
cyanobacterium is invariably a N2-fixing species that provides the plant with fixed
nitrogen (Bergman et al., 2008). The cyanobacterium in the symbiosis has a low
photosynthetic activity and is in turn supplemented by the plant partner with sugars
(Adams and Duggan, 2008; Meeks, 2009). Although fructose and glucose 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, however, 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 (N) deprivation some
cells differentiate into N2-fixing heterocysts (Flores and Herrero, 2010). Nostoc
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 establishes de novo associations (Adams and Duggan, 2008;
Meeks and Elhai, 2002). 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
non-vascular
plant
of
the
division
Anthocerotophyta (hornworts) whose gametophytes support the growth of
microcolonies of Nostoc sp., 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), originally
isolated from a root section of the cycad Macrozamia sp. (Rippka et al., 1979), can
establish symbiosis with A. punctatus (Enderlin and Meeks, 1983).
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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 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 and other cyanobacteria are fructose, glucose, and sucrose
(Rippka et al., 1979). Fructose or glucose transport activity has been reported for
some Nostoc strains (Beauclerk and Smith, 1978; Schmetterer and Flores, 1986),
including Nostoc filaments isolated from a symbiotic association (Black et al., 2002).
These sugars have also been shown to support nitrogenase activity in a Nostoc sp.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
(MFS)
glucose
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 fructose uptake (Flores and Schmetterer, 1986). The second is an
ABC-type fructose 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 homologues 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 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 transportrelated cluster and analyzed their ability to establish symbiosis with A. punctatus.
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RESULTS
Sugar transporters
In the genome of N. punctiforme, a cluster of genes is present encoding
homologues to the A. variabilis ABC-type fructose transporter (Frt), the
Synechocystis glucose 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 homologues (frtA1 and frtA2), one frtB and one frtC (Fig. 1). Immediately
downstream of frtC is glcP, and about 1 kb downstream of the latter, 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 Fig. 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 wildtype strain (ATCC 29133) or in 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 (Suppl. Fig. 1).
Filaments of N. punctiforme strains ATCC 29133 and ATCC 29133-S (UCD
153) grown in BG110 + NH4+ medium showed uptake of [14C]fructose and
[14C]glucose linearly for at least one hour, with strain UCD 153 exhibiting somewhat
higher activities than the wild type (Suppl. Fig. 2). 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 to their respective parental strains. Strains
in which the frt genes were removed (CSME1A, CSME1B, and CSME-S1A) showed
very low uptake of fructose (Table 1), consistent with the notion that Frt is a fructose
transporter. Fructose uptake was also impaired (42 to 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 fructose
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uptake, consistent with the idea that this protein is a sugar porin. Strains CSMES13A and CSME-S13B, in which only GlcP was removed, showed a low or an
intermediate rate of fructose uptake, respectively. Whereas the results with strain
CSME-S13B may imply that GlcP also mediates fructose transport, the results with
CSME-S13A most likely indicate a further effect of C.K3, which in the latter strain
was inserted in opposite orientation to the genes (see below).
Strains CSME-S13A and CSME-S13B showed very low levels of glucose
uptake (Table 1), consistent with the notion that GlcP is a glucose transporter, and
strains CSME-S12A and CSME-S12B were impaired in glucose uptake to a similar
extent as in fructose 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 glucose uptake. On the other hand, the effect on glucose 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 glucose uptake was very low.
When C.K3 was in the same orientation as the genes in the gene cluster, 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. Northern blot analysis with probes of either
glcP or oprB showed an exceptionally high expression of these genes in strain
CSME1B (Suppl. Fig. 3), which can account for the observed increase in glucose
uptake (Table 1). 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 fructose uptake;
high expression from C.K3 could result in negative effects on transcription, mRNA
stability or translation.
The results obtained indicate that the putative ABC-type Frt transporter does
mediate fructose uptake, the identified GlcP is a glucose permease, and the OprBlike porin is needed for optimal uptake of both fructose and glucose. Results of
growth analysis with sugar supplementation were consistent with the transport data.
As shown in Fig. 2, N. punctiforme grew heterotrophically better with fructose than
with glucose, which corroborates the results of Summers et al. (1995), but growth
did not take place in the CSME1A strain that is hampered in 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; see Fig. 2), of this strain
specifically on glucose. Finally, strain CSME11 could not grow with glucose, but
showed some growth with fructose, consistent with its lack of activity of glucose
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transport and its partial activity of fructose transport. Heterotrophic growth that was
more robust with fructose than with glucose was also observed with strain UCD 153
(not shown).
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 green
fluorescent protein (GFP) to the 7th codon of frtA1 was prepared in two constructs.
One DNA fragment covering the intergenic region between hrmE and frtA1 and
another fragment extending backwards 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 CSMES19) showed strong GFP fluorescence in vegetative cells of ammonium-grown
filaments (Fig. 3A to C) or of filaments incubated in the absence of a source of
combined nitrogen (Fig. 3E, F), although some variability in fluorescence was
observed between cells. GFP fluorescence was very high in akinetes (Fig. 3A, B, 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 heterocysts, GFP fluorescence was extremely low or null (Fig.
3E, F). High GFP expression in vegetative cells and lack of expression in
heterocysts was corroborated in strain CSME-S18 (not shown), which carries only
the region upstream from frtA1, indicating that the regulated expression of the frt
genes in heterocysts vs. 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, 2005); therefore, HrmR, the homologue of FrtR, does not appear to be
involved in transcriptional regulation of the frt genes.
Symbiotic phenotype
Hormogonia are the infection units of N. punctiforme in 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 (not
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shown). The symbiotic phenotype of the mutants was investigated by adding each
mutant to an A. punctatus culture that had been growing on N-free medium
supplemented with 28 mM glucose for 7-10 days. Glucose was added for optimal
growth of A. punctatus under low light (31.5 µmol m-2 s-1). The infection frequency
was scored after two weeks of co-culture 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 1. N. punctiforme mutants that showed a very low activity of
glucose transport or a low activity of both glucose and fructose transport did not
infect A. punctatus. Thus, all the glcP mutants and the frt mutants in which C.K3 was
in opposite orientation to the sugar transport genes did not infect the plant. In
contrast, strain CSME1B that showed a very low fructose 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 four-week test for those mutants that had been observed to infect the plant. The
glcP-defective strain CSME13A, which showed the lowest glucose 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 free-living state autotrophically, photoheterotrophically or
dark heterotrophically using glucose, fructose, ribose or, weakly, sucrose (Rippka et
al., 1979; Summers et al., 1995). We have shown that glucose uptake is mediated by
the GlcP MFS permease and fructose uptake by the FrtA1-FrtA2-FrtB-FrtC ABC-type
transporter. The homologous glucose permease from Synechocystis 6803 might also
mediate fructose 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 fructose uptake.
This could imply that GlcP may also aid fructose 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 glucose uptake in strains
CSME1A and CSME-S1A, that interrupt the frt genes by antiparallel insertion of the
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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 5’ 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 uptake of both fructose and glucose independently of the sense in which the
C.K3 cassette is inserted, we infer that the encoded protein facilitates uptake of both
sugars. This is consistent with the identification based on homology of this protein as
OprB, a 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 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
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
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
hours, HIF elicited an early (<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 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
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
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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 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 appears, therefore, 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 hormogoniumrepressing factor, HRF (Meeks, 2006), which also appears to be present in the plant
exudate containing HIF (Campbell et al., 2008). This would imply 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 glucose uptake (Table 1) and increased heterotrophic growth with glucose
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 glucose uptake by
the symbiont is consistent with the results of a previous study in which a significant
fraction of glucose taken up by infected tissue of a Gunnera-Nostoc symbiosis was
observed to correspond to assimilation by the cyanobacterium (Black et al., 2002). In
another study, N-deprived Gunnera plants were found to accumulate high levels of
glucose and fructose. However, Nostoc 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 fructose or any other sugar that can be assimilated
by N. punctiforme, such as sucrose or ribose, has a role in nutrition of the
cyanobacterial symbiont. Decreased fructose uptake as in strain CSME1B did not
affect by itself symbiotic performance, but the high activity of glucose uptake taking
place in this strain might compensate for the missing fructose uptake.
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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 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 colonies, while the latter is seen in the regions of highest nitrogen fixation
activity (for example see, 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 (Khamar et
al., 2010; Nilsson et al., 2006; Rasmussen et al., 1996). It has been also suggested
that various sugars, including glucose and fructose, 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 act in this way,
then one might expect to find a regulatory connection between the hrm genes and
the sugar transporters. Indeed, the 5’ region of frtA1 and hrmI share a conserved
sequence (TGAAAACTGTAGTTT) that in hrmI is located between the -10 and -35
RNA polymerase sigma subunit recognition sequences where binding of a putative
regulatory protein would inhibit transcription; such binding has not been
experimentally determined (Meeks 2005). Finally, because a high cellular C to N
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 (Meeks, 2005). 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.
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MATERIALS AND METHODS
Bacterial strains and growth conditions
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 [N-tris(hydroxymethyl)methyl-2aminoethanesulfonic acid]-NaOH buffer [pH 7.5]) at 30°C in the light (25 μmol m-2 s-1)
in shaken (100 rpm) liquid cultures or on medium solidified with 1% Difco agar. For
the mutants described below, neomycin [Nm] was used in liquid media at 10 μg ml-1
and in solid media at 25 μg ml-1. For heterotrophic growth assays, 20 μl of N.
punctiforme strain cell suspensions were placed on plates containing either 5 mM
fructose or 5 mM glucose, in addition to BG110/NH4+/TES/agar, and incubated in
darkness. Escherichia coli strain DH5α, used for plasmid constructions, and strains
HB101 and ED8654, used for conjugations with N. punctiforme strains, were grown
in LB 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 Suppl. Table
1). 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 Nmr and Kmr (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 localization of frtA1 gene expression, a
fragment including the first seven codons of Npun_R5327 and 988 bp upstream of
the same gene (including 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
14
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(Olmedo-Verd et al., 2006) producing a translational fusion of the gfp-mut2 gene
(Cormarck et al., 1996) to the Npun_R5327 5’ 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
(2144 bp upstream from Npun_R5327, including also the hrmE gene and its
upstream region, extending 27 nt 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 3' 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 ml of E. coli strain HB101 carrying helper/methylation plasmid pRL623
and a cargo plasmid was harvested at exponential phase (approx. OD750= 1.0) and
mixed with an equal amount of E. coli ED8654 carrying conjugative plasmid pRL443,
giving a final volume of approximately 150 µl. After one hour of mating, 100 µl of N.
punctiforme culture, at a concentration of 175 μg chlorophyll a (Chla) ml-1, was added
to the E. coli mixture, which was then plated on IMMOBILON NC Millipopre filters
placed on top of BG110/NH4+/TES/LB5% plates. After a 24-h incubation at 30°C under
low light (5 μmol m-2 s-1), 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 μmol
m-2 s-1). Finally the membranes were transferred to BG110/NH4+/TES /Nm25 plates
and incubated at 25°C, normal light, until Nm-resistant colonies appeared. For gene
inactivations, double recombinants were selected from single recombinants by
growth on plates containing 5% sucrose, 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 Suppl. Fig. 1.
Transport assays
For
14
C-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 μg Chla ml-1. The assay
was started by adding 0.2 ml of a sugar solution containing either 1.1 mM glucose
and 15 μM [14C]glucose (300 Ci mol-1, Perkin Elmer) or 1.1 mM fructose and 15 μM
[14C]fructose (300 Ci mol-1, Perkin Elmer) to a 2-ml cell suspension. The cultures
15
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
were incubated at 30°C at a light intensity of 85 μmol m-2 s-1 and 0.5-ml samples
were filtered, using 0.45-μm-poresize 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.
Microscopy
N. punctiforme ATCC 29133 and strains CSME18 and CSME19 were grown
in liquid media with or without a source of combined nitrogen. Samples from these
cultures were analyzed in a Leica TCS SP5 confocal laser-scanning 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 µmol m-2 s-1), and also supplemented with 0.5%
(w/v) glucose for optimal growth. To reconstitute the symbiotic associations, A.
punctatus tissue was first incubated in glucose-supplemented basal medium minus
ammonium nitrate for 7-10 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 Chla content of 60 µg 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 co-culturing for 2 weeks.
ACKNOWLEDGMENTS
We thank Antonia Herrero for generous support and Jeff Elhai and Wan-Ling Chiu for
critically reading the manuscript.
16
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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FIGURE LEGENDS
Figure 1. Structure of the sugar transport genomic region in Nostoc punctiforme and
mutants generated in this work. Open reading frame numbers (Npun_) and proposed
gene names are indicated for each gene. Two putative pseudogenes, Npun_R5321
and Npun_R5322 (not shown in the picture), 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 Nostoc
punctiforme strains bearing those constructs are indicated. Note that in each case
the gene cassette replaced a DNA fragment in the chromosome. In the lower right
corner, the DNA fragments used to promote gfp expression in Nostoc punctiforme
strains carrying the corresponding constructs in plasmid pRL25C are depicted. The
sizes of the genes and intergenic regions are shown.
Figure. 2. Heterotrophic growth with sugars of Nostoc punctiforme strain ATCC
29133 (WT) and some sugar transport mutants (CSME# strains). Filament samples
were spotted in BG110 + NH4+ solid medium supplemented with 5 mM fructose (Frc)
or 5 mM glucose (Glc) and incubated in the dark for 2 weeks.
Figure. 3. Expression of GFP in Nostoc punctiforme strain CSME-S19 bearing an
frtA1-gfp-mut2 translational fusion. Filaments of CSME-S19 were grown in BG110 +
NH4+ medium (A to C) and, after washing with BG110 medium (lacking any source of
combined nitrogen), incubated in BG110 medium for the indicated times (D to 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) are shown. No green
fluorescence was observed with the parental strain UCD 153 (not shown). Filaments
in A, B and C are composed mostly of vegetative cells. Brackets in A, B and G
indicate an akinete or akinete rows. The filament enclosed in a square in C is an
hormogonium, and the filament in D is in part differentiating into an hormogonium
(note enclosure). Arrowheads in E and F point to heterocysts (which lack
autofluorescence) that are found next to vegetative cells. Size bars are 10 µm (E, F,
G), 20 µm (A) or 40 µm (B, C, D).
Figure. 4. Co-culture of Anthoceros punctatus with examples of Nostoc punctiforme
sugar transport mutants. (Top) Flasks containing 15 to 20 g of fresh plant tissue in
nitrogen-free growth medium were inoculated with the indicated cyanobacterial
21
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
strains. (Bottom)
Micrographs of tissue from the flasks above, in which some
cyanobacterial colonies are highlighted by circles. Photographs were taken after four
weeks of co-culture.
Figure. 5. Schematic of the Nostoc-plant symbiotic interaction. The cyanobacterium
forms many more heterocysts (double-circled cells in the scheme) in symbiosis than
in the free-living state. In the A. punctatus symbiosis, nitrogen 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 glucose
and of any other possible substrate of the permease in the symbiosis.
22
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Table 1. Fructose and glucose uptake and symbiotic performance in Nostoc punctiforme
parental strains and mutants.
Strain
Fructose uptakea
-1
Glucose uptakea
-1
-1
(nmol [mg Chl] min )
Rate ± St. Dev. (n)
Symbiotic
-1
phenotypeb
(nmol [mg Chl] min )
%
Rate ± St. Dev. (n)
%
11.6 ± 7.1 (3)
100
21.2 ± 10.2 (4)
100
+
CSME1A
0.3 ± 0.1 (3)
3
1.3 ± 0.3 (4)
6
-
CSME1B
0.9 ± 0.1 (3)
8
125.6 ± 47.6 (4)
592
+
CSME11
4.9 ± 3.3 (3)
42
1.2 ± 0.4 (4)
6
-
UCD 153
38.0 ± 18.8 (8)
100
44.0 ± 10.4 (8)
100
+
CSME-S1A
0.4 ± 0.1 (3)
1
1.7 ± 0.6 (4)
4
-
CSME-S12A
29.0 ± 7.5 (6)
76
22.3 ± 5.4 (5)
51
+
CSME-S12B
24.1 ± 10.1 (5)
63
32.1 ± 7.7 (5)
73
+
CSME-S13A
10.1 ± 8.5 (6)
27
0.6 ± 0.4 (5)
1
-
CSME-S13B
17.8 ± 2.2 (4)
47
0.9 ± 0.02 (3)
2
-
ATCC 29133
(a)
Transport assays were carried out with 100 µM substrate for 40 to 60 min as described in
Materials and methods. Each number is the mean and standard deviation of the results
from the number of experiments performed with independent cultures indicated in
parenthesis.
(b)
The symbiotic phenotype was scored as positive if the presence of symbiotic colonies in
plant tissue was observed after two weeks of co-culture. For 15 to 20 g of fresh plant tissue,
about 400-600 colonies were observed for ATCC 29133 and its positive derivatives and 60100 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 four weeks of co-culture.
23
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Fig. 1 CSME‐S12A CSME‐S13A CSME‐S12B CSME‐S13B oprB glcP R5320 frtC R5323 1614bp 1401bp 992bp frtB R5324 R5325 999bp 100bp 1557bp frtA2 frtA1 hrmE R5326 R5327 R5328 996bp 897bp 1023bp 276bp 38bp CSME1B CSME11 gfp CSME1A CSME‐S1A gfp 556bp 1006bp 2162bp CSME‐S18 CSME‐S19 Fig. 1. Structure of the sugar transport genomic region in Nostoc punc7forme and mutants generated in this work. Open reading frame numbers (Npun_) and proposed gene names are indicated for each gene. Two putaOve pseudogenes, Npun_R5321 and Npun_R5322 (not shown in the picture), can be found between Npun_R5320 and Npun_R5323. The site of inserOon and the orientaOon of the C.K3 gene casseSe (gray arrows) are shown for the different constructs, and the names of the Nostoc punc7forme strains bearing those constructs are indicated. Note that in each case the gene casseSe replaced a DNA fragment in the chromosome. In the lower right corner, the DNA fragments used to promote gfp expression in Nostoc punc7forme strains carrying the corresponding constructs in plasmid pRL25C are depicted. The sizes of the genes and intergenic regions are shown. Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Fig. 2 WT
1A
1B
11
Frc
Glc
Fig. 2. Chemoheterotrophic growth of Nostoc punctiforme strain ATCC 29133 (WT) and
some sugar transport mutants (CSME# strains). Filament samples were spotted in
BG110 + NH4+ solid medium supplemented with 5 mM fructose (Frc) or 5 mM glucose
(Glc) and incubated in the dark for 2 weeks.
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Fig. 3 (A) CSME‐S19 (NH4+) (B) CSME‐S19 (NH4+) (C) CSME‐S19 (NH4+) (D) CSME‐S19 (24 h –N) (E) CSME‐S19 (25 h –N) (F) CSME‐S19 (40 h –N) (G) CSME‐S19 (40 h –N) Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Fig. 3. Expression of GFP in Nostoc punctiforme strain CSME-S19 bearing an frtA1-gfpmut2 translational fusion. Filaments of CSME-S19 were grown in BG110 + NH4+ medium
(A to C) and, after washing with BG110 medium (lacking any source of combined
nitrogen), incubated in BG110 medium for the indicated times (D to 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) are shown. No green fluorescence was observed with the
parental strain UCD 153 (not shown). Filaments in A, B and C are composed mostly of
vegetative cells. Brackets in A, B and G indicate an akinete or akinete rows. The
filament enclosed in a square in C is an hormogonium, and the filament in D is in part
differentiating into an hormogonium (note enclosure). Arrowheads in E and F point to
heterocysts (which lack autofluorescence) that are found next to vegetative cells. Size
bars are 10 µm (E, F, G), 20 µm (A) or 40 µm (B, C, D).
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Fig. 4 CSME-S13A
CSME-S12A
Fig. 4. Co-culture of Anthoceros punctatus with examples of Nostoc punctiforme sugar
transport mutants. (Top) Flasks containing 15 to 20 g of fresh plant tissue in nitrogenfree growth medium were inoculated with the indicated cyanobacterial strains. (Bottom)
Micrographs of tissue from the flasks above, in which some cyanobacterial colonies are
highlighted by circles. Photographs were taken after four weeks of co-culture.
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Fig. 5 Plant
C
N
Nostoc
(up to 60% heterocysts)
Fig. 5. Schematic of the Nostoc-plant symbiotic interaction. The cyanobacterium forms
many more heterocysts (double-circled cells in the scheme) in symbiosis than in the
free-living state. In the A. punctatus symbiosis, nitrogen fixed in the heterocysts is
transferred in the form of ammonia to the plant by an unknown molecular mechanism.
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 glucose and of any other possible substrate of the
permease in the symbiosis.