BACKGROUND
biodiesel produced by photosynthetic microorganisms, such as eukaryotic algae and cyanobacteria, provides a promising alternative to reduce our reliance on fossil fuels.
biodiesel can be produced either directly by these organisms or from their biomass.
this fuel is carbon neutral, renewable and its use requires minimal changes in the current structure of fuel delivery and consumption.
however, the relatively low productivity and the high cost of harvesting the biomass present mayor limitations for commercialization of cyanobacteria-derived biodiesels  <cit> .
these limitations have been mainly addressed by using a rational approach  <cit> .
the rational approach involves designing strains with enhanced biodiesel precursor production  by modifying and/or introducing known metabolic pathways.
the most common modifications include the introduction of a heterologous thioesterase and removal of the endogenous acyl-acp synthetase.
the introduced thioesterase is capable of hydrolysing the acyl-acp molecule and thus releases the fatty acid, the major precursor of biodiesel  <cit> .
the acyl-acp synthetase can reactivate free fatty acids by attaching them to the acp-molecule  <cit> .
by introducing a thioesterase in combination with disrupting the endogenous acyl-acp synthetase, one can significantly enhance fatty acid production and secretion in cyanobacteria  <cit> .
however, despite some success, the reported fatty acid yields are still not sufficient for large-scale production.
this is partly due to the fact that the bulk of the fatty acids remain within the cell.
changing the cell structure in such a way that the cells can auto-flocculate or elongate might help to harvest the remaining biomass more efficiently  <cit> .
since the rational approach is guided by existing knowledge about the cellular processes involved in fatty acid biosynthesis and/or transport, it can limit strain improvement due to missing information on key intermediates, regulators, competing pathways etc.
therefore, combining the rational approach with random approaches  may result in the identification of novel genes involved in efficient biodiesel precursor production.
random mutagenesis has been successfully used in cyanobacteria to isolate filamentous  <cit>  and grazing resistant mutants  <cit>  as well as to identify environmentally responsive genes  <cit>  and genes involved in polyhydroxybutyrate synthesis  <cit> .
furthermore, this approach was previously applied to isolate genes involved in fatty acid production in escherichia coli  <cit> .
however, genes identified by hoover and colleagues  in e. coli lack obvious orthologous genes in cyanobacteria.
moreover, to our knowledge, random mutagenesis has never been applied to isolate lipid-overproducing mutants in cyanobacteria.
in this study we used synechococcus elongatus pcc  <dig> which is amenable to genetic modification and has a fully sequenced genome.
this strain produces the major biodiesel precursors and lacks the ability to synthesize phb   <cit> .
these properties make s. elongatus pcc  <dig> a good model strain to isolate mutants with an enhanced fatty acid production.
the construction and analysis of a transposon generated mutant library, resulted in the identification of a gene that directly or indirectly affects fatty acid and hydrocarbon production.
moreover, this gene, named era, is also involved in determining the cellular morphology of pcc  <dig>  to our knowledge this gene has never been analysed in cyanobacteria.
RESULTS
isolation of a putative fatty acid overproducing mutant by random mutagenesis
to obtain a mutant library, we haven chosen to construct a transposon library using plasmid prl1063a, which contains a tn <dig> transposon with a kanamycin resistance marker, promoterless luxab reporter genes and an origin of replication which functions in e. coli, but not in pcc  <dig>  this library, contained approximately  <dig> individual mutants, was subsequently screened for mutants with beneficial phenotypes such as filamentous and auto-flocculating growth, higher lipid content and phenotypic stability.
out of the initial  <dig> mutants a single strain was selected for further study based on its  elongated cell morphology,  enhanced fluorescence after staining with the lipophilic dye nile red and  phenotypic stability.
the selected mutant, 2a <dig>  was both phenotypically and genotypically stable for a period of at least  <dig> months .
this mutant showed a different colony morphology compared to pcc  <dig> .
2a <dig> has a round, slightly elevated colony morphology with irregular shaped edges on plate  while pcc  <dig> is round, raised with an entire smooth edge .
phase contrast microscopy revealed that cultures of mutant 2a <dig> contained a heterogeneous mix of short cells similar in size to pcc  <dig> and long elongated cells some more than  <dig> times the length of pcc  <dig> .figure  <dig>
macro  and microscopic morphology  of wild-type pcc  <dig>  and mutant 2a <dig> .
macroscopic morphology was visualized after growth on plate using a stereomicroscope.
microscopic morphology was visualized by phase-contrast microscopy.
scale bars represent  <dig> μm.
example of an elongated cell is indicated with an arrow .
we also used flow cytometry to determine cellular morphology  of pcc  <dig> and 2a <dig> stained with nile red  or unstained .
stained cells are very similar to unstained cells in respects to their cellular size , however the complexity  after staining with nile red is slightly, but significantly higher.
comparison of cells of pcc  <dig>  and 2a <dig> , furthermore indicates that cells of 2a <dig> are both larger in overall size and complexity than pcc  <dig> figure  <dig>
side scatter  versus front scatter  as determined using flow cytometry.
pcc  <dig>  and mutant 2a <dig>  grown in liquid media for  <dig> hours either unstained  or stained  with the lipophilic dye nile red.
the largest differences between pcc  <dig> and 2a <dig> where observed when comparing the yellow fluorescence  of nile red stained cells.
in general, unstained cells showed a low amount of yellow fluorescence compared to both red and green fluorescence .
cells stained with nile red showed an increase of yellow fluorescence  and 2a <dig> showed a higher increase in yellow fluorescence even at comparable cellular size  than pcc  <dig> figure  <dig>
yellow fluorescence versus front scatter  as determined using flow cytometry.
pcc  <dig>  and mutant 2a <dig>  grown in liquid media for  <dig> hours either unstained  or stained  with the lipophilic dye nile red.
transposon insertion site
to identify the insertion site of the transposon, genomic dna was isolated, digested, re-ligated and transformed into e. coli dh5α.
plasmid dna was extracted from colonies that acquired antibiotic resistance and restriction patterns were compared to identify additional transposition events.
based on their distinct restriction patterns, dna of two isolated plasmids was sequenced.
both sequences showed the insertion of the transposon into the  <dig> prime region of gene synpcc7942_ <dig> annotated as a gtp-binding protein era.
the insertion caused the formation of an early stop codon leading to a truncated protein lacking the last  <dig> amino acids.
the gtp-binding protein era of pcc  <dig> contains both a gtp binding and atp hydrolysing domain at its n terminus and a type  <dig> k homology  domain at its c-terminus .
in pcc  <dig>  this gene is the first gene in an operon containing a total of  <dig> genes  <cit> .
verification genotype-phenotype relationship
to identify the genotype, phenotype relationship of mutant 2a <dig> and to elucidate the function of the gtp-binding protein era in pcc  <dig>  several rational approaches were used.
to test the effect of overexpression of the gtp-binding protein era in pcc <dig>  a construct containing an iptg inducible promoter followed by a ribosomal binding site and the full-length wild type era gene was made .
insertion of this construct into the neutral site  <dig> from pcc  <dig>  <cit>  was obtained after transformation, resulting in strain se:era.
full segregation was verified by pcr amplification.
as a negative control, a similar construct lacking the ribosomal binding site and era gene  was inserted into the neutral site  <dig> from pcc  <dig> .
to reconstruct the original phenotype, the plasmid containing the transposon flanked by genomic dna isolated from 2a <dig> was reinserted into the genome of pcc  <dig> after inactivating the transposase by introducing a frame shift mutation.
this mutant showed the correct insertion and was fully segregated .
this mutant was complemented by introducing the overexpression construct pns3:era, resulting in strain se:∆era + era and as a negative control the construct lacking the ribosomal binding site and era gene was inserted into neutral site  <dig>  mutant se:∆era.
in addition, a knockout construct was made in which the coding region of the era gene was replaced by a km resistance cassette.
mutants obtained revealed an elongated phenotype .
however after subcultivation, these initial mutants either lost viability, or the wild type phenotype was restored.
growth
all strains show similar specific growth rates as measured using od <dig> .
the growth rates of mutant strains were  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  h− <dig> for se:era, se:Δera and se:Δera + era respectively.
these specific growth rates were not significantly different from that of pcc  <dig> .
the final biomass of mutant strains at time of harvesting was also not significantly different compared to pcc  <dig> figure  <dig>
cellular growth of wild type pcc  <dig>  strain se:era, se:∆era and se:∆era + era.
growth was measured at  <dig> hour intervals using the optical density at  <dig> nm.
error bars represent the standard deviation of four replicates.
cellular morphology: length distribution during growth
to monitor changes in cell size, aliquots from cultures were examined using a phase contrast microscopy .figure  <dig>
distribution of cell lengths in liquid cultures of pcc  <dig> and mutant se:era, se:∆era and se:∆era + era after  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  hours.
lengths of cells of each strain was determined at  <dig> hour intervals by taking pictures using a phase contrast microscope and using imagej to determine their cell size.
strain se:era, containing an additional copy of era, is significantly smaller at the start of the experiment  compared to wt with an median of  <dig>  and  <dig>  μm, respectively.
at time points  <dig>   <dig>  and  <dig> hours  both pcc  <dig> and se:era have a similar median cell length of  <dig> ,  <dig>  and  <dig> - <dig>  μm , respectively.
at  <dig> hours, the median cell length of pcc  <dig> is significantly larger compared to se:era .
at the time of harvesting  the cell length of pcc  <dig> elongated further to  <dig>  μm while cells from se:era had a median cell length of  <dig>  μm.
strain se:∆era has a significantly larger median cell size compared to pcc  <dig> at all time points.
se:∆era had a median cell length of  <dig>  μm at the time of inoculation .
this cell length decreased in the first  <dig> hours of growth to  <dig>  μm.
after this time point the cell length increase to  <dig> ,  <dig> ,  <dig>  and  <dig>  μm at  <dig>   <dig>   <dig> and  <dig> hours, respectively.
strain se:∆era + era has a significantly reduced cell length compared to se:∆era, but is larger than pcc  <dig> at all time points .
cellular morphology: microscopy
differential interference microscopic analysis of the mutant and wild-type strains revealed that strain pcc  <dig>  se:era and se:∆era:era appear very similar to each other in cell size at  <dig> hours .
this result is similar to the results obtained by measuring the cell size .
furthermore, septum formation occurs in a symmetric manner for all these mutants.figure  <dig>
morphology of pcc  <dig> , mutant se:era , se:∆era  and se:∆era + era  grown in liquid media for  <dig> hours.
scale bars represent  <dig> μm.
the site of septa formation are indicated with arrows.
mutant se:∆era however has both elongated cells and cell similar in size to pcc  <dig>  constrictions can occur similar to pcc  <dig>  at the middle of the cell .
however, constrictions are often observed more towards the cellular pole  or sometimes multiple times within a single cell .
furthermore, full separation of the daughter cells does also not always occur leading to the formation of cells arrested at the pre-divisional two-cell stage.
fatty acid and hydrocarbon profile pcc 7942
pcc  <dig> contains a mixture of saturated and unsaturated fatty acids ranging from c <dig> to c <dig>  palmitic  and palmitoleic acid  represent the main intracellular fatty acids of pcc  <dig> .
together these fatty acids represent approximately 89%  of the total fatty acids .
the remainder mainly consists of saturated and unsaturated c <dig> and c <dig> fatty acids followed by trace amounts of odd chain c15: <dig>  c17: <dig> and c17: <dig> figure  <dig>
quantitative measurement of intracellular fatty acids in pcc  <dig>  strain se:era, se:∆era and se:∆era + era.
metabolites indicated with an asterisk * are significantly different  from those of strain pcc  <dig>  error bars represent the standard deviation of four replicates.
pcc  <dig> is able to synthesize both alkanes and alkenes .
the alkanes synthesized by pcc  <dig> are mostly heptadecane , followed by pentadecane  and hexadecane .
the only alkene that we detected was 8-heptadecene at levels comparable to those of hexadecane.figure  <dig>
quantitative measurement of intracellular hydrocarbons in pcc  <dig>  strain se:era, se:∆era and se:∆era + era.
metabolites indicated with an asterisk * are significantly different  from those of strain pcc  <dig>  error bars represent the standard deviation of four replicates.
fatty acid and hydrocarbon profile se:era
strain se:era, which contains an additional era gene inserted at neutral site  <dig>  has a significant increase in the total amount of fatty acids .
the amount of fatty acids increased to  <dig>  6 ±  <dig>  μg/mg in se:era versus  <dig>  ±  <dig>  μg/mg dw in pcc <dig> .
this increase in fatty acids is mostly due to a significant rise in the amount of c16: <dig> , c18: <dig>  and to a far lesser extend c17: <dig> .
together they account for 68% of the increase in total fatty acids, 42%, 26% and 1% for c16: <dig>  c18: <dig> and c17: <dig> respectively.
no differences were observed in the amount of hydrocarbons compared to pcc  <dig> .
fatty acid and hydrocarbon profile se: Δera
strain se:Δera, containing the inactivated transposon, contains a similar amount  of total fatty acids as pcc  <dig>  .
nevertheless, there are some significant changes in the amount of individual fatty acids.
this strain has an 162% increase in the amount of c14: <dig> and an 93% increase in c14: <dig> compared to pcc <dig>  furthermore, it has a 39% reduction in the amount of c18: <dig> and a 21% reduction in the amount of c18: <dig> versus pcc  <dig>
this strain is the only strain with significant changes in the hydrocarbon profile .
it contains a higher amount of pentadecane and 8-heptadecene compared to pcc  <dig>  25% and 76% respectively.
furthermore, it has a substantial reduction  in the amount of heptadecane.
fatty acid and hydrocarbon profile se: Δera + era
strain se:Δera + era, containing the inactivated transposon complemented ectopically by era, has an almost identical amount of total fatty acids  compared to pcc  <dig>  .
the only fatty acid, which showed a significant difference, is myristic acid .
this fatty acid was increased by 31% compared to pcc  <dig>
the hydrocarbons were similar compared to pcc  <dig> .
discussion
the era gene is essential for s. elongatus pcc 7942
in this study we generated a transposon library of s. elongatus pcc  <dig> in order to isolate mutants with beneficial biodiesel production traits, including enhanced fatty acid and hydrocarbon content.
the transposon mutant 2a <dig> was selected based on  increased staining with the lipophilic dye nile red ,  elongated cell morphology  and  phenotypic stability.
the transposon of mutant 2a <dig> was inserted into the  <dig> prime region of the synpcc7942_ <dig>  gene.
due to this insertion, a premature stop codon was introduced into the kh-domain of this protein.
the kh domain is involved in rna binding in e. coli and the gtp binding domain regulates its activity  <cit> .
moreover, all gtp-binding protein era homologs that have been studied to date for membrane binding activity  are associated with the cytoplasmic membrane  <cit> .
this activity is most likely also linked with the kh domain which is well conserved in all gtp-binding protein era homologs including that of s. elongatus pcc  <dig>  <cit> .
the era gene is the first gene of an operon containing a total of  <dig> genes.
these include  <dig> genes encoding hypothetical proteins  and  <dig> that encode a trna-met, an iojap-like protein and the gtp-binding protein era itself .
genes, which are grouped into operons together in multiple species, likely have similar functions even if transcribed individually in other species  <cit> .
this suggests that era has a similar function in cyanobacteria as in e. coli in linking nutrient stress, to other cellular processes such as protein synthesis , photosynthesis and cell division  <cit> .
to study whether the phenotype of the transposon insertion is  caused by a possible effect on the expression of downstream genes, we attempted to create a knockout of the era gene.
we prevented transcription of all downstream genes by replacing the era gene with an antibiotic cassette flanked by two transcriptional terminators.
unfortunately, several transformation experiments with this cassette did not result in the isolation of the expected mutant, suggesting that one or several of the genes downstream of era are essential.
however, subsequent introduction of a knockout cassette lacking the transcriptional terminators resulted in the isolation of a transient knockout mutant of era.
after subcultivation of this knockout mutant, cells either failed to grow or the wild type phenotype was restored.
restoration of the wild type phenotype was associated with the removal of the resistance cassette from the era gene.
these results indicates that era might also be an essential gene in pcc  <dig> as was previously shown for e. coli  <cit> .
moreover, we found that the transient mutant also showed elongated cells and problems in septa formation.
in addition, the deletion mutation was fully complemented by the introduction of an ectopic era gene .
we further verified the phenotype-genotype relationship by a) overexpressing era b) recreating the original transposon mutant, and c) complementing the recreated transposon mutant with an ectopic copy of era.
gtp-binding protein era is involved in lipid and hydrocarbon production of pcc 7942
analysis of an era overexpression strain, se:era, showed a significantly higher amount of fatty acids compared to pcc  <dig> .
these results demonstrate the importance of the gtp-binding protein era for the production of fatty acids in s. elongatus pcc  <dig>
to further verify the role of era in biodiesel precursor production, we reintroduced the inactive transposon in pcc  <dig> and complemented the resulted se:∆era mutant with an ectopic copy of era.
strain se:∆era does not show a significant difference in the total amount of fatty acids, compared to pcc <dig> .
however, because this strain has significantly longer cells , there are fewer cells per mg of dry weight compared to pcc  <dig>  these results indicate that there is a significantly higher amount of lipids per cell, supporting the original nile red results.
moreover, several individual fatty acids are significantly different from pcc  <dig> , suggesting that era plays a role in fatty acid synthesis in this strain.
complementation of se:∆era with the ectopically integrated era gene  fully restored the fatty acid profile to pcc  <dig> , supporting the previous observation that era is responsible for the changes in fatty acid profile.
in se:era we did not observe any significant changes in hydrocarbon production.
however, we found significant changes in hydrocarbon production in se:∆era, which were restored when complemented with era , suggesting that era is also involved in hydrocarbon metabolism.
rational strain improvement for biodiesel precursor production of strain pcc  <dig> has been attempted in the past by the introduction of thioesterase, acetyl-coa carboxylase and ribulose- <dig> -bisphosphate carboxylase/oxygenase.
however, these modifications failed to increase the net fa production  <cit> .
moreover, identified targets using rna-seq to enhance the strain productivity of pcc  <dig> led only to minor improvements in fa production  <cit> .
however, our results show a substantial increase in fatty acid production without causing negative effects on biomass accumulation, suggesting that the gtp-binding protein era is a new promising gene target for further strain improvement for the production of fatty acids .
gtp-binding protein era is important for cell size of pcc 7942
several studies in e. coli showed that overexpression of era up to 5% of total cellular protein does not alter cellular growth, camp levels or protein production  <cit> .
this is possibly due to the fact that the activity of the gtp-binding protein era is regulated by binding of gtp  <cit> .
however, we observed a significantly effect on cellular size during late exponential growth of pcc  <dig> containing an additional era gene.
although the effect on cell size is subtle and can easily be missed during microscopic examination, cells of se:era are significantly smaller than those of pcc  <dig> .
to our knowledge, this is the first report demonstrating that overexpression of era causes a change in cell size.
depletion of era in e. coli causes a decrease in growth, loss of viability, septum formation and elongated cells  <cit> .
in the mutant se:∆era, containing the inactivated transposon, we did observe an effect on septum formation and cell size.
however, this mutant did not show a difference in growth rate nor viability compared to pcc  <dig>  analysis of the phenotype of se:∆era suggests that the expression of era might be reduced to a level which is slightly lower than that required for normal cell division  <cit> .
introduction of an ectopic era in strain se:∆era resulted in restoring the formation of septa in the middle of a cell, but not entirely the cell length.
while more than 96% of all the cells were within the same length range as pcc  <dig>  some cells were up to 26% larger than wt.
however, the median length of these cells was 31% smaller compared to strain se:∆era.
these results suggest the insertion in era itself and/or one or more downstream genes is responsible for the elongated cell morphology.
the fact that introduction of an additional era gene did not fully complement the se:∆era mutant, might have several reasons.
first of all, the expression of the era gene used for complementation is not regulated by its native promoter.
for example, it has been shown that expression of era is coupled to cellular growth  in e. coli  <cit> .
another possibility is that downstream genes might be responsible for part of the phenotype and/or presence of a truncated era interferes with the complementation.
CONCLUSIONS
it was previously shown that era plays a role in many cellular processes including ribosome assembly and cell cycle regulation  <cit> .
this protein has been well studied in diverse bacterial species, but never in cyanobacteria.
in this report we showed that era is an essential gene for s. elongatus pcc  <dig>  in this strain, the gtp-binding protein era is involved in hydrocarbon and fatty acid metabolism as well as the determination of cell size.
a possible explanation for the observed changes in fatty acid and hydrocarbons is the role that era plays in coupling nutrient stress, cell division and photosynthesis.
in a normal situation era delays cell division and synthesis of fatty acids, causing cells to become longer and synthesize less proteins and fatty acids.
in the era overexpression strain , this regulation is slightly disrupted causing almost no change in cellular size  and a relative increased fatty acid synthesis.
we propose that the era protein can be used as a novel target for improving biofuel production traits.
it would be interesting to evaluate the effect of overexpression of era in combination with other genetic modifications, for example in a strain expressing a thioesterase and containing a knockout of the acyl-acp synthetase.
furthermore, the question remains open whether overproduction of the gtp-binding protein era also influences cellular size and fatty acid/hydrocarbon content in other bacteria.

 biodiesel produced by photosynthetic microorganisms, such as eukaryotic algae and cyanobacteria, provides a promising alternative to reduce our reliance on fossil fuels. biodiesel can be produced either directly by these organisms or from their biomass. this fuel is carbon neutral, renewable and its use requires minimal changes in the current structure of fuel delivery and consumption. however, the relatively low productivity and the high cost of harvesting the biomass present mayor limitations for commercialization of cyanobacteria-derived biodiesels. these limitations have been mainly addressed by using a rational approach. the rational approach involves designing strains with enhanced biodiesel precursor production by modifying and/or introducing known metabolic pathways. the most common modifications include the introduction of a heterologous thioesterase and removal of the endogenous acyl-acp synthetase. the introduced thioesterase is capable of hydrolysing the acyl-acp molecule and thus releases the fatty acid, the major precursor of biodiesel. the acyl-acp synthetase can reactivate free fatty acids by attaching them to the acp-molecule. by introducing a thioesterase in combination with disrupting the endogenous acyl-acp synthetase, one can significantly enhance fatty acid production and secretion in cyanobacteria. however, despite some success, the reported fatty acid yields are still not sufficient for large-scale production. this is partly due to the fact that the bulk of the fatty acids remain within the cell. changing the cell structure in such a way that the cells can auto-flocculate or elongate might help to harvest the remaining biomass more efficiently. since the rational approach is guided by existing knowledge about the cellular processes involved in fatty acid biosynthesis and/or transport, it can limit strain improvement due to missing information on key intermediates, regulators, competing pathways etc. therefore, combining the rational approach with random approaches may result in the identification of novel genes involved in efficient biodiesel precursor production. random mutagenesis has been successfully used in cyanobacteria to isolate filamentous and grazing resistant mutants as well as to identify environmentally responsive genes and genes involved in polyhydroxybutyrate synthesis. furthermore, this approach was previously applied to isolate genes involved in fatty acid production in escherichia coli. however, genes identified by hoover and colleagues in e. coli lack obvious orthologous genes in cyanobacteria. moreover, to our knowledge, random mutagenesis has never been applied to isolate lipid-overproducing mutants in cyanobacteria. in this study we used synechococcus elongatus pcc which is amenable to genetic modification and has a fully sequenced genome. this strain produces the major biodiesel precursors and lacks the ability to synthesize phb. these properties make s. elongatus pcc a good model strain to isolate mutants with an enhanced fatty acid production. the construction and analysis of a transposon generated mutant library, resulted in the identification of a gene that directly or indirectly affects fatty acid and hydrocarbon production. moreover, this gene, named era, is also involved in determining the cellular morphology of pcc to our knowledge this gene has never been analysed in cyanobacteria.  isolation of a putative fatty acid overproducing mutant by random mutagenesis to obtain a mutant library, we haven chosen to construct a transposon library using plasmid prl1063a, which contains a tn transposon with a kanamycin resistance marker, promoterless luxab reporter genes and an origin of replication which functions in e. coli, but not in pcc this library, contained approximately individual mutants, was subsequently screened for mutants with beneficial phenotypes such as filamentous and auto-flocculating growth, higher lipid content and phenotypic stability. out of the initial mutants a single strain was selected for further study based on its elongated cell morphology, enhanced fluorescence after staining with the lipophilic dye nile red and phenotypic stability. the selected mutant, 2a was both phenotypically and genotypically stable for a period of at least months. this mutant showed a different colony morphology compared to pcc. 2a has a round, slightly elevated colony morphology with irregular shaped edges on plate while pcc is round, raised with an entire smooth edge. phase contrast microscopy revealed that cultures of mutant 2a contained a heterogeneous mix of short cells similar in size to pcc and long elongated cells some more than times the length of pcc.figure macro and microscopic morphology of wild-type pcc and mutant 2a. macroscopic morphology was visualized after growth on plate using a stereomicroscope. microscopic morphology was visualized by phase-contrast microscopy. scale bars represent μm. example of an elongated cell is indicated with an arrow. we also used flow cytometry to determine cellular morphology of pcc and 2a stained with nile red or unstained. stained cells are very similar to unstained cells in respects to their cellular size, however the complexity after staining with nile red is slightly, but significantly higher. comparison of cells of pcc and 2a, furthermore indicates that cells of 2a are both larger in overall size and complexity than pcc figure side scatter versus front scatter as determined using flow cytometry. pcc and mutant 2a grown in liquid media for hours either unstained or stained with the lipophilic dye nile red. the largest differences between pcc and 2a where observed when comparing the yellow fluorescence of nile red stained cells. in general, unstained cells showed a low amount of yellow fluorescence compared to both red and green fluorescence. cells stained with nile red showed an increase of yellow fluorescence and 2a showed a higher increase in yellow fluorescence even at comparable cellular size than pcc figure yellow fluorescence versus front scatter as determined using flow cytometry. pcc and mutant 2a grown in liquid media for hours either unstained or stained with the lipophilic dye nile red. transposon insertion site to identify the insertion site of the transposon, genomic dna was isolated, digested, re-ligated and transformed into e. coli dh5α. plasmid dna was extracted from colonies that acquired antibiotic resistance and restriction patterns were compared to identify additional transposition events. based on their distinct restriction patterns, dna of two isolated plasmids was sequenced. both sequences showed the insertion of the transposon into the prime region of gene synpcc7942_ annotated as a gtp-binding protein era. the insertion caused the formation of an early stop codon leading to a truncated protein lacking the last amino acids. the gtp-binding protein era of pcc contains both a gtp binding and atp hydrolysing domain at its n terminus and a type k homology domain at its c-terminus. in pcc this gene is the first gene in an operon containing a total of genes. verification genotype-phenotype relationship to identify the genotype, phenotype relationship of mutant 2a and to elucidate the function of the gtp-binding protein era in pcc several rational approaches were used. to test the effect of overexpression of the gtp-binding protein era in pcc a construct containing an iptg inducible promoter followed by a ribosomal binding site and the full-length wild type era gene was made. insertion of this construct into the neutral site from pcc was obtained after transformation, resulting in strain se:era. full segregation was verified by pcr amplification. as a negative control, a similar construct lacking the ribosomal binding site and era gene was inserted into the neutral site from pcc. to reconstruct the original phenotype, the plasmid containing the transposon flanked by genomic dna isolated from 2a was reinserted into the genome of pcc after inactivating the transposase by introducing a frame shift mutation. this mutant showed the correct insertion and was fully segregated. this mutant was complemented by introducing the overexpression construct pns3:era, resulting in strain se:∆era + era and as a negative control the construct lacking the ribosomal binding site and era gene was inserted into neutral site mutant se:∆era. in addition, a knockout construct was made in which the coding region of the era gene was replaced by a km resistance cassette. mutants obtained revealed an elongated phenotype. however after subcultivation, these initial mutants either lost viability, or the wild type phenotype was restored. growth all strains show similar specific growth rates as measured using od. the growth rates of mutant strains were  ± ,  ±  and  ±  h− for se:era, se:Δera and se:Δera + era respectively. these specific growth rates were not significantly different from that of pcc. the final biomass of mutant strains at time of harvesting was also not significantly different compared to pcc figure cellular growth of wild type pcc strain se:era, se:∆era and se:∆era + era. growth was measured at hour intervals using the optical density at nm. error bars represent the standard deviation of four replicates. cellular morphology: length distribution during growth to monitor changes in cell size, aliquots from cultures were examined using a phase contrast microscopy.figure distribution of cell lengths in liquid cultures of pcc and mutant se:era, se:∆era and se:∆era + era after,,,, and hours. lengths of cells of each strain was determined at hour intervals by taking pictures using a phase contrast microscope and using imagej to determine their cell size. strain se:era, containing an additional copy of era, is significantly smaller at the start of the experiment compared to wt with an median of and μm, respectively. at time points and hours both pcc and se:era have a similar median cell length of, and - μm, respectively. at hours, the median cell length of pcc is significantly larger compared to se:era. at the time of harvesting the cell length of pcc elongated further to μm while cells from se:era had a median cell length of μm. strain se:∆era has a significantly larger median cell size compared to pcc at all time points. se:∆era had a median cell length of μm at the time of inoculation. this cell length decreased in the first hours of growth to μm. after this time point the cell length increase to,, and μm at and hours, respectively. strain se:∆era + era has a significantly reduced cell length compared to se:∆era, but is larger than pcc at all time points. cellular morphology: microscopy differential interference microscopic analysis of the mutant and wild-type strains revealed that strain pcc se:era and se:∆era:era appear very similar to each other in cell size at hours. this result is similar to the results obtained by measuring the cell size. furthermore, septum formation occurs in a symmetric manner for all these mutants.figure morphology of pcc, mutant se:era, se:∆era and se:∆era + era grown in liquid media for hours. scale bars represent μm. the site of septa formation are indicated with arrows. mutant se:∆era however has both elongated cells and cell similar in size to pcc constrictions can occur similar to pcc at the middle of the cell. however, constrictions are often observed more towards the cellular pole or sometimes multiple times within a single cell. furthermore, full separation of the daughter cells does also not always occur leading to the formation of cells arrested at the pre-divisional two-cell stage. fatty acid and hydrocarbon profile pcc 7942 pcc contains a mixture of saturated and unsaturated fatty acids ranging from c to c palmitic and palmitoleic acid represent the main intracellular fatty acids of pcc. together these fatty acids represent approximately 89% of the total fatty acids. the remainder mainly consists of saturated and unsaturated c and c fatty acids followed by trace amounts of odd chain c15: c17: and c17: figure quantitative measurement of intracellular fatty acids in pcc strain se:era, se:∆era and se:∆era + era. metabolites indicated with an asterisk * are significantly different from those of strain pcc error bars represent the standard deviation of four replicates. pcc is able to synthesize both alkanes and alkenes. the alkanes synthesized by pcc are mostly heptadecane, followed by pentadecane and hexadecane. the only alkene that we detected was 8-heptadecene at levels comparable to those of hexadecane.figure quantitative measurement of intracellular hydrocarbons in pcc strain se:era, se:∆era and se:∆era + era. metabolites indicated with an asterisk * are significantly different from those of strain pcc error bars represent the standard deviation of four replicates. fatty acid and hydrocarbon profile se:era strain se:era, which contains an additional era gene inserted at neutral site has a significant increase in the total amount of fatty acids. the amount of fatty acids increased to 6 ±  μg/mg in se:era versus  ±  μg/mg dw in pcc. this increase in fatty acids is mostly due to a significant rise in the amount of c16:, c18: and to a far lesser extend c17:. together they account for 68% of the increase in total fatty acids, 42%, 26% and 1% for c16: c18: and c17: respectively. no differences were observed in the amount of hydrocarbons compared to pcc. fatty acid and hydrocarbon profile se: Δera strain se:Δera, containing the inactivated transposon, contains a similar amount of total fatty acids as pcc. nevertheless, there are some significant changes in the amount of individual fatty acids. this strain has an 162% increase in the amount of c14: and an 93% increase in c14: compared to pcc furthermore, it has a 39% reduction in the amount of c18: and a 21% reduction in the amount of c18: versus pcc this strain is the only strain with significant changes in the hydrocarbon profile. it contains a higher amount of pentadecane and 8-heptadecene compared to pcc 25% and 76% respectively. furthermore, it has a substantial reduction in the amount of heptadecane. fatty acid and hydrocarbon profile se: Δera + era strain se:Δera + era, containing the inactivated transposon complemented ectopically by era, has an almost identical amount of total fatty acids compared to pcc. the only fatty acid, which showed a significant difference, is myristic acid. this fatty acid was increased by 31% compared to pcc the hydrocarbons were similar compared to pcc. discussion the era gene is essential for s. elongatus pcc 7942 in this study we generated a transposon library of s. elongatus pcc in order to isolate mutants with beneficial biodiesel production traits, including enhanced fatty acid and hydrocarbon content. the transposon mutant 2a was selected based on increased staining with the lipophilic dye nile red, elongated cell morphology and phenotypic stability. the transposon of mutant 2a was inserted into the prime region of the synpcc7942_ gene. due to this insertion, a premature stop codon was introduced into the kh-domain of this protein. the kh domain is involved in rna binding in e. coli and the gtp binding domain regulates its activity. moreover, all gtp-binding protein era homologs that have been studied to date for membrane binding activity are associated with the cytoplasmic membrane. this activity is most likely also linked with the kh domain which is well conserved in all gtp-binding protein era homologs including that of s. elongatus pcc. the era gene is the first gene of an operon containing a total of genes. these include genes encoding hypothetical proteins and that encode a trna-met, an iojap-like protein and the gtp-binding protein era itself. genes, which are grouped into operons together in multiple species, likely have similar functions even if transcribed individually in other species. this suggests that era has a similar function in cyanobacteria as in e. coli in linking nutrient stress, to other cellular processes such as protein synthesis, photosynthesis and cell division. to study whether the phenotype of the transposon insertion is caused by a possible effect on the expression of downstream genes, we attempted to create a knockout of the era gene. we prevented transcription of all downstream genes by replacing the era gene with an antibiotic cassette flanked by two transcriptional terminators. unfortunately, several transformation experiments with this cassette did not result in the isolation of the expected mutant, suggesting that one or several of the genes downstream of era are essential. however, subsequent introduction of a knockout cassette lacking the transcriptional terminators resulted in the isolation of a transient knockout mutant of era. after subcultivation of this knockout mutant, cells either failed to grow or the wild type phenotype was restored. restoration of the wild type phenotype was associated with the removal of the resistance cassette from the era gene. these results indicates that era might also be an essential gene in pcc as was previously shown for e. coli. moreover, we found that the transient mutant also showed elongated cells and problems in septa formation. in addition, the deletion mutation was fully complemented by the introduction of an ectopic era gene. we further verified the phenotype-genotype relationship by a) overexpressing era b) recreating the original transposon mutant, and c) complementing the recreated transposon mutant with an ectopic copy of era. gtp-binding protein era is involved in lipid and hydrocarbon production of pcc 7942 analysis of an era overexpression strain, se:era, showed a significantly higher amount of fatty acids compared to pcc. these results demonstrate the importance of the gtp-binding protein era for the production of fatty acids in s. elongatus pcc to further verify the role of era in biodiesel precursor production, we reintroduced the inactive transposon in pcc and complemented the resulted se:∆era mutant with an ectopic copy of era. strain se:∆era does not show a significant difference in the total amount of fatty acids, compared to pcc. however, because this strain has significantly longer cells, there are fewer cells per mg of dry weight compared to pcc these results indicate that there is a significantly higher amount of lipids per cell, supporting the original nile red results. moreover, several individual fatty acids are significantly different from pcc, suggesting that era plays a role in fatty acid synthesis in this strain. complementation of se:∆era with the ectopically integrated era gene fully restored the fatty acid profile to pcc, supporting the previous observation that era is responsible for the changes in fatty acid profile. in se:era we did not observe any significant changes in hydrocarbon production. however, we found significant changes in hydrocarbon production in se:∆era, which were restored when complemented with era, suggesting that era is also involved in hydrocarbon metabolism. rational strain improvement for biodiesel precursor production of strain pcc has been attempted in the past by the introduction of thioesterase, acetyl-coa carboxylase and ribulose- -bisphosphate carboxylase/oxygenase. however, these modifications failed to increase the net fa production. moreover, identified targets using rna-seq to enhance the strain productivity of pcc led only to minor improvements in fa production. however, our results show a substantial increase in fatty acid production without causing negative effects on biomass accumulation, suggesting that the gtp-binding protein era is a new promising gene target for further strain improvement for the production of fatty acids. gtp-binding protein era is important for cell size of pcc 7942 several studies in e. coli showed that overexpression of era up to 5% of total cellular protein does not alter cellular growth, camp levels or protein production. this is possibly due to the fact that the activity of the gtp-binding protein era is regulated by binding of gtp. however, we observed a significantly effect on cellular size during late exponential growth of pcc containing an additional era gene. although the effect on cell size is subtle and can easily be missed during microscopic examination, cells of se:era are significantly smaller than those of pcc. to our knowledge, this is the first report demonstrating that overexpression of era causes a change in cell size. depletion of era in e. coli causes a decrease in growth, loss of viability, septum formation and elongated cells. in the mutant se:∆era, containing the inactivated transposon, we did observe an effect on septum formation and cell size. however, this mutant did not show a difference in growth rate nor viability compared to pcc analysis of the phenotype of se:∆era suggests that the expression of era might be reduced to a level which is slightly lower than that required for normal cell division. introduction of an ectopic era in strain se:∆era resulted in restoring the formation of septa in the middle of a cell, but not entirely the cell length. while more than 96% of all the cells were within the same length range as pcc some cells were up to 26% larger than wt. however, the median length of these cells was 31% smaller compared to strain se:∆era. these results suggest the insertion in era itself and/or one or more downstream genes is responsible for the elongated cell morphology. the fact that introduction of an additional era gene did not fully complement the se:∆era mutant, might have several reasons. first of all, the expression of the era gene used for complementation is not regulated by its native promoter. for example, it has been shown that expression of era is coupled to cellular growth in e. coli. another possibility is that downstream genes might be responsible for part of the phenotype and/or presence of a truncated era interferes with the complementation.  it was previously shown that era plays a role in many cellular processes including ribosome assembly and cell cycle regulation. this protein has been well studied in diverse bacterial species, but never in cyanobacteria. in this report we showed that era is an essential gene for s. elongatus pcc in this strain, the gtp-binding protein era is involved in hydrocarbon and fatty acid metabolism as well as the determination of cell size. a possible explanation for the observed changes in fatty acid and hydrocarbons is the role that era plays in coupling nutrient stress, cell division and photosynthesis. in a normal situation era delays cell division and synthesis of fatty acids, causing cells to become longer and synthesize less proteins and fatty acids. in the era overexpression strain, this regulation is slightly disrupted causing almost no change in cellular size and a relative increased fatty acid synthesis. we propose that the era protein can be used as a novel target for improving biofuel production traits. it would be interesting to evaluate the effect of overexpression of era in combination with other genetic modifications, for example in a strain expressing a thioesterase and containing a knockout of the acyl-acp synthetase. furthermore, the question remains open whether overproduction of the gtp-binding protein era also influences cellular size and fatty acid/hydrocarbon content in other bacteria. 
based on the screening results, we selected a single mutant, which has an insertion in the gene encoding for the gtp-binding protein era. the gtp-binding protein era has never been studied in cyanobacteria and proved to be an essential gene for s. elongatus pcc we also found that this protein is important for hydrocarbon and fatty acid metabolism as well as determination of the cell size in pcc our results suggest that the gtp-binding protein era can be used as a novel target for further improvement of biofuel precursors production. in this study we created a transposon library of synechococcus elongatus pcc in order to identify novel gene targets for enhanced fatty acid and hydrocarbon production. ectopic complementation of this mutation fully restored the hydrocarbon production profile to that of wild-type and partially restored the fatty acid production. overexpression of era caused a reduction in the cell size in the late exponential phase of growth and an increase in the total amount of intracellular fatty acids. reintroduction of the inactivated transposon caused a significant increase in the cellular length as well as changes in the amounts of individual hydrocarbons and fatty acids. biofuelscyanobacteriagtp-binding protein erafatty acidshydrocarbonssynechococcus elongatus pcc 7942issue-copyright-statement© the author 2015 moreover, the cellular size was significantly smaller than that of the inactivated transposon mutant. the transposon library was subsequently screened for desirable traits using macro- and microscopic observations as well as staining with the lipophilic dye nile red. biodiesel production using cyanobacteria is a promising alternative to fossil fuels.
 biodiesel production using cyanobacteria is a promising alternative to fossil fuels. in this study we created a transposon library of synechococcus elongatus pcc in order to identify novel gene targets for enhanced fatty acid and hydrocarbon production. the transposon library was subsequently screened for desirable traits using macro- and microscopic observations as well as staining with the lipophilic dye nile red.  based on the screening results, we selected a single mutant, which has an insertion in the gene encoding for the gtp-binding protein era. we subsequently verified the phenotype-genotype relation by overexpression, reintroducing and complementing the mutation. overexpression of era caused a reduction in the cell size in the late exponential phase of growth and an increase in the total amount of intracellular fatty acids. reintroduction of the inactivated transposon caused a significant increase in the cellular length as well as changes in the amounts of individual hydrocarbons and fatty acids. ectopic complementation of this mutation fully restored the hydrocarbon production profile to that of wild-type and partially restored the fatty acid production. moreover, the cellular size was significantly smaller than that of the inactivated transposon mutant.  the gtp-binding protein era has never been studied in cyanobacteria and proved to be an essential gene for s. elongatus pcc we also found that this protein is important for hydrocarbon and fatty acid metabolism as well as determination of the cell size in pcc our results suggest that the gtp-binding protein era can be used as a novel target for further improvement of biofuel precursors production. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords biofuelscyanobacteriagtp-binding protein erafatty acidshydrocarbonssynechococcus elongatus pcc 7942issue-copyright-statement© the author 2015 