- Research article
- Open Access
Genome analysis of the freshwater planktonic Vulcanococcus limneticus sp. nov. reveals horizontal transfer of nitrogenase operon and alternative pathways of nitrogen utilization
BMC Genomics volume 19, Article number: 259 (2018)
Many cyanobacteria are capable of fixing atmospheric nitrogen, playing a crucial role in biogeochemical cycling. Little is known about freshwater unicellular cyanobacteria Synechococcus spp. at the genomic level, despite being recognised of considerable ecological importance in aquatic ecosystems. So far, it has not been shown whether these unicellular picocyanobacteria have the potential for nitrogen fixation. Here, we present the draft-genome of the new pink-pigmented Synechococcus-like strain Vulcanococcus limneticus.
sp. nov., isolated from the volcanic Lake Albano (Central Italy).
The novel species Vulcanococcus limneticus sp. nov. falls inside the sub-cluster 5.2, close to the estuarine/marine strains in a maximum-likelihood phylogenetic tree generated with 259 marker genes with representatives from marine, brackish, euryhaline and freshwater habitats. V.limneticus sp. nov. possesses a complete nitrogenase and nif operon. In an experimental setup under nitrogen limiting and non-limiting conditions, growth was observed in both cases. However, the nitrogenase genes (nifHDK) were not transcribed, i.e., V.limneticus sp. nov. did not fix nitrogen, but instead degraded the phycobilisomes to produce sufficient amounts of ammonia. Moreover, the strain encoded many other pathways to incorporate ammonia, nitrate and sulphate, which are energetically less expensive for the cell than fixing nitrogen. The association of the nif operon to a genomic island, the relatively high amount of mobile genetic elements (52 transposases) and the lower observed GC content of V.limneticus sp. nov. nif operon (60.54%) compared to the average of the strain (68.35%) support the theory that this planktonic strain may have obtained, at some point of its evolution, the nif operon by horizontal gene transfer (HGT) from a filamentous or heterocystous cyanobacterium.
In this study, we describe the novel species Vulcanococcus limneticus sp. nov., which possesses a complete nif operon for nitrogen fixation. The finding that in our experimental conditions V.limneticus sp. nov. did not express the nifHDK genes led us to reconsider the actual ecological meaning of these accessory genes located in genomic island that have possibly been acquired via HGT.
The reduction of atmospheric dinitrogen gas (N2) to ammonia (NH3) is a very energetically expensive process because of the high stability of the dinitrogen molecule, due to the triple bond between the two nitrogen atoms . At the same time nitrogen is a key element for primary productivity in the ocean  and nitrogen fixation is a fundamental process in several aquatic environments [3, 4]. A minimum set of six nif conserved genes is required in bacteria for nitrogen fixation. Three code for structural and catalytic components (nifHDK) and three (nifENB) for components involved in the biosynthesis of FeMoco enzymes, which are involved in dinitrogenase activation . Transcription of nifHDK can be used as a proxy for nitrogen fixation levels .
Diazotrophs are microorganisms capable of converting dinitrogen into ammonia . Cyanobacteria are the only oxygen producing microorganisms able to perform nitrogen fixation . Typically, cyanobacterial nitrogenases are organized in different operons, nifB-fdxN-nifSU, nifHDK, nifENXW and nifVZT . The nitrogenase complex is irreversibly damaged by oxygen, thus diazotrophic microorganisms have developed different strategies to ensure the success of this process. The filamentous heterocystous cyanobacteria differentiate a specialized nitrogen-fixing cell, the heterocyst, that constitutes an anaerobic site well protected from external oxygen by a thick membrane. Colonial filamentous non-heterocystous cyanobacteria (e.g. Planktothrix serta), which lack heterocysts, segregate nitrogen fixation and photosynthesis both spatially and temporally [8, 9]. Non-heterocystous diazotrophic unicellular cyanobacteria (e.g. Cyanothece, Synechococcus) evolved a temporal separation strategy, fixing nitrogen during the night and performing photosynthesis during the day, thus separating oxygenic PSII activity from the nitrogenase complex .
These last kind of unicellular cyanobacteria have been recognized to be important in marine systems and are potentially abundant enough to significantly contribute to oceanic nitrogen fixation [11, 12]. The presence of a nif operon in the cyanobacterial ancestor is controversial: on the one hand it could be acquired by horizontal gene transfer (HGT) after the origin of this clade, probably as consequence of the “fixed nitrogen crisis” between the Archean and Proterozoic era, which led to a lack of available nitrogen in the upper ocean promoting acquisition of nif genes from a heterotrophic prokaryote via HGT . Alternatively, a more recent paper  proposed that the nif genes were present in the cyanobacterial ancestor, repeatedly lost and gained again within the cyanobacterial phylum.
In the light of the central role of nitrogen fixation in biogeochemical cycles, it is crucial to study the nif operon in terms of presence, phylogenetic relationships and functionality in strains within the cyanobacterial clades. The ubiquitous unicellular cyanobacteria, Synechococcus, is a polyphyletic genus  currently being reclassified  and is recognized as being of ecological importance in aquatic ecosystems . Yet it has often been neglected regarding nitrogen fixation and only few data are found in the literature on this issue. Only two sequenced Synechococcus from Yellowstone hot spring mat  were reported to contain the nif operon and were described as nitrogen fixing. Certain strains showed nitrogenase activity only under anaerobic conditions , while others were active also under microaerobic conditions. This is the case of the Synechococcus SF1 isolated from the blades of Sargassum fluitans which protects the nitrogenase complex by consuming excess oxygen through Uptake Hydrogenase activity [20, 21]. Another interesting strategy was used by Synechococcus RF-1, isolated from a rice field, capable of rhythmic nitrogen-fixing activity . The marine Synechococcus strains Miami BG43511 and BG43522 developed a temporal regulation . Nevertheless, there is no evidence for the occurrence of planktonic freshwater Synechococcus-like species with the potential for nitrogen fixation, nor for the activity of nif genes by RNA transcript quantification.
In order to better understand the possible contribution of freshwater picocyanobacteria to nitrogen fixation we studied Vulcanococcus limneticus sp. nov. (formerly Synechococcus LL) isolated from a volcanic lake in central Italy, previously sequenced at draft genome level, finding it positive for the nif operon. Thus: i) we analyzed the sequences of the genes within the nif operon and the phylogenetic relationships between this cluster and others from Synechococcus and other cyanobacteria strains, ii) we measured nifHDK transcripts under nitrogen limitation conditions and iii) we analyzed the genome to find other ways for this strain to use nitrogen.
Results and discussion
Phenotypic and genomic features of Vulcanococcus limneticus sp. nov.
In this work we present the genome of V.limneticus sp. nov., a novel planktonic strain isolated from the volcanic Lake Albano in central Italy (41°44′47.5″N, 12°40′14.3″E) (Additional file 1: Table S1) . This strain, previously described as Synechococcus LL and used in experiments [24, 55], is composed by phycoerythrin-rich cells (Additional file 2: Fig. S2). The genome has a total size of 3,548,882 bp and the GC content of the strain is 68.35%.
Phylogenomics of the planktonic Vulcanococcus limneticus sp. nov.
Several studies in the past few years reported that Synechococcus and Cyanobium sub-clusters 5.2 and 5.3 comprise marine, brackish, euryhaline and freshwater strains [15, 25, 26], whilst sub-cluster 5.1 solely contains marine representatives [27,28,29]. Additionally, reports based on 16S rRNA genes of freshwater strains have revealed the presence of 13 non-marine clusters inside the 5.3 and 5.2 sub-clusters [24, 30]. Other phylogenomic studies have showed that Synechococcus is not a monophyletic group , and only recently new classifications and novel genera within the traditionally considered Synechococcus clade have been proposed . Based on our phylogenomic results evaluated with PhyloPlAn tool  with a total of 259 universal markers, we noted that the planktonic Vulcanococcus limneticus sp. nov. falls inside the sub-cluster 5.2 close to the estuarine/marine strains Synechococcus CB0205 and CB0101 (isolated from Chesapeake Bay) (Fig. 1). Recent phylogenomic studies led to genus proposal of Magnicoccus for CB0205 and CB0101 strains , and it appears that our strain affiliates close to these representatives from this new genus. Nonetheless, it presents less than 79% of average nucleotide identity (ANI) and less than 72% of average amino acid identity (AAI) with closest strains as GFB01 and CB0201, hence is the first representative of a novel picocyanobacterial genus. Genome-to-genome DNA hybridization (GGDH), ANI and AAI values with closest species are shown in Additional file 3: Table S2. The enormous diversity seen in sub-cluster 5.2 opens new perspectives for picocyanobacterial evolution, once that studies on a novel pigment gene in the Baltic Sea raised the possibility that picocyanobacteria of the subcluster 5.2 originated in freshwater sources (; P.Sánchez-Baracaldo et al. unpublished).
Nitrogenase operon in the freshwater strain Vulcanococcus limneticus sp. nov
Here, for the first time, we show the presence of nitrogenase operon in the new planktonic freshwater picocyanobacteria V.limneticus sp. nov. As shown in Fig. 2, V.limneticus sp. nov. exhibits a complete nif operon with nifHDK catalytic subunits, nifBSU, nifENXW biosynthetic proteins and the additional subunits hesB, hesA, nifV, nifZ, fixU, the transcriptional regulator of nif operon XRE (xenobiotic response element), the molybdenum transporter modAB and the ferredoxins fdxB and fdxH. The latter was described as important for the maximum nitrogenase activity in heterocystous cyanobacteria as its inactivation delays nitrogen fixation , but it is not essential for their growth . Only nifP was absent from this strain. We compared the structure of the nif operon with some representatives of cyanobacteria and provided a phylogeny of the nifHDK concatamer among different molybdenum, vanadium and iron nitrogenases from different bacterial phyla representatives as previously described [35,36,37,38]. Compared to the rest of cyanobacteria (Fig. 2 and Fig. 3) the heterocystous Anabaena variabilis and Nostoc display a completely different organization of the nif operon with some subunits located elsewhere in their genomes. V.limneticus sp. nov. contains a slightly different nif structure compared with the rest of the other strains, although the closest gene organization is observed in the Yellowstone Hot Springs Synechococcus JA-3-3Ab and JA-2-3Ba , where the structure of nifBSUHDKVZ is maintained except for the insertion of the ferredoxin fdxB in these strains between nifB and nifS subunits. The gene organization compared to anaerobic cyanobacteria also displays different arrangements (Fig. 3), especially compared to the unicellular Cyanothece sp. PCC 7425 . The novel. V.limneticus sp. nov. nitrogenase affiliates with a cluster of molybdenum cyanobacterial nitrogenases (Fig. 4), although it appears quite distant from heterocystous strains like Anabaena and Nostoc and forms a different branch from Synechococcus JA-3-3Ab and JA-2-3Ba, and other strains like Planktothrix serta PCC8927, Phormidium ambiguum IAMM-71 and Chroococcidiopsis thermalis PCC 7203, Oscillatoria sp. PCC 6506, and Neosynechococcus sphagnicola sy1. In general, except for the high similarity (90–100%) of the nif operon for Synechococcus JA-3-3Ab and JA-2-3Ba, we observed low levels of genetic identity (from 50 to 80%) despite the well conserved synteny of the different subunits between the different cyanobacteria tested, confirming an enormous genetic diversity of nitrogenases in the cyanobacterial phylum.
Evidence of HGT of the nif operon in V.Limneticus sp. nov.
A previous study carried out in the cyanobacterium Microcoleus chthonoplastes showed acquisition of its nif operon by HGT . We found some genomic features inside the genome of this planktonic strain that could provide evidence for a HGT of its nif operon. Following metagenomic fragment recruitment methods described in other publications for freshwater Synechococcus  we noted the highest abundance of V.limneticus sp. nov. in two Amazon lakes from which metagenomic studies were carried out . This strain was not detected significantly (above species level, > 95% of ANI) in other available freshwater metagenomes from all over the world. It must be noted the lack of metagenomes of volcanic lakes. As depicted from Additional file 4: Fig. S1, we observed that V.limneticus sp. nov. genome is fully covered both above the species level (95%) and between 80 and 95% of identity values, which confirms that V.limneticus sp. nov. clones from the same species (> 95%) are present in these tropical Amazon lakes together with closely and distantly related species (80–95%). The regions of low coverage in these metagenomes, also called genomic islands, were associated to LPS biosynthesis, genetic mobile elements or other phage defense systems, as previously described in marine Synechococcus . However, we noted that nif operon was also associated with a genomic island, being absent from the majority of the Synechococcus population and apparently rare in the tropical Lake Ananá and Mancapuru Great lake. The GC content of the contig containing the nif operon (26,472 bp and 60.54%) also contrasts with the GC content of the strain (68.35%). The genes flanking the nif operon contain an average GC content of 65–67%, whilst the majority of nif genes present lower values from 56 to 61% and give top BLAST hits to heterocystous, filamentous and other Cyanobacteria as Leptolyngbya, Lyngbya, Phormidium, Oscillatoria, Microcoleus, Kamptonema, Calothrix, Pseudoanabaena or Tolypothrix species. Moreover, we noted a relatively high amount of transposases and mobile elements in V.limneticus sp. nov. (52), mostly at the beginning and at the end of the different contigs, which confirms that this strain is genetically prepared for gene transfer events. These features may reinforce the theory that this planktonic strain may have obtained, at some point of its evolution, the nif operon by HGT from a filamentous or heterocystous cyanobacterium.
Expression of nifHDK genes
To estimate the expression level of the nifHDK genes of V.limneticus sp. nov.we measured RNA transcripts using real time PCR in two experimental conditions: in the presence and absence of nitrogen. V.limneticus sp. nov.was able to grow in both conditions (Additional file 2: Fig. S2). However, the RNA transcript quantification of the nifHDK genes gave negative results or comparable to those of the non-reverse transcribed RNA (NORT) (Additional file 5: Table S3). Considering these results, we can assume that nifHDK genes were not expressed in our experimental conditions. We must also consider that the 16S rRNA gene was always expressed with threshold cycle values between 18.7–19.4 for all the replicates with the CT values for the NORT negative or between 31.7–34.8 in presence and absence of nitrogen (Additional file 5: Table S3). Thus, although V.limneticus sp. nov. is able to grow in nitrogen limiting conditions, we might speculate that this strain is not able to perform nitrogen fixation, at least in our experimental conditions (not excluding the possibility of the nitrogen fixation capability by V.limneticus sp. nov. in anaerobic conditions). We acknowledge the need to test the transcript quantification of the nifHDK genes in other laboratory conditions (e.g. in anaerobic conditions, during other hours of the diel cycle) to make our results more comprehensive. We recognized some similarities with the filamentous, non-heterocystous cyanobacterium Microcoleus chthonoplastes that possesses nif genes but is unable to express nitrogenase in culture .
Possible alternative nitrogen sources for V.Limneticus sp. nov. under nitrogen limiting conditions
When V.limneticus sp. nov. is under nutrient limitation, it accumulates glycogen (observed in our –N culture, Additional file 6: Fig. S3) and the phycobilisomes are specifically and rapidly degraded, in a process known as bleaching or chlorosis . We noticed that when keeping the cultures for a longer time-span (one month), the flasks of the treatment without nitrogen bleached while the treatment with nitrogen were of an intense pink colour (Fig. 5). The cells in the –N treatment showed a strong aggregation and a lack of yellow fluorescence under blue excitation (Fig. 5). This phenomenon suggests a loss of the phycobilin pigments, likely indicating a use of the pigments as nitrogen reserve. The degradation of phycobilisomes under nitrogen starvation requires the activity of alanine dehydrogenase (ald gene) producing sufficient amounts of ammonia . This gene was found in the V.limneticus sp. nov. genome pointing to other mechanisms of nitrogen acquisition. The V.limneticus sp. nov. genome harbored multiple mechanisms to incorporate ammonia into the cell. For instance, we detected the ubiquitous and widespread ammonia assimilatory pathway via glutamine synthetase, ferredoxin-dependent glutamate synthetase, glutamine amidotransferase, nitrogen regulatory protein pII and amt transporters. Cyanate hydrolysis has been reported in marine Synechococcus and Prochlorococcus under nitrogen depleted conditions . Interestingly, we detected the cyanate hydratase (CynS) that catalyses the transformation of cyanate into ammonia and CO2, which could provide an evolutionary advantage of V.limneticus sp. nov. under nitrogen limitation in Lake Albano. We also observed the nitrate and nitrite ammonification pathway as an alternative source to get ammonia; we detected a nitrate assimilatory reductase (which catalyses the transformation of nitrate to nitrite) and an ammonia ferredoxin oxidoreductase or nitrite reductase (which catalyses the transformation of nitrite into ammonia). Together with these enzymes, we also found two nitrate ABC transporters and four nitrate/sulfonate ABC transporters that could also be used for the uptake of sulfonates and alkanesulfonates.
Other metabolic features of V.Limneticus sp. nov.
It was reported that in nitrogen-fixing cyanobacteria hydrogen is synthesized as a by-product of nitrogenase activity and is further oxidized by a hydrogenase [7, 8]. There are two types of hydrogenases in cyanobacteria: an uptake hydrogenase (HupSL) which catalyses H2 consumption and is present in almost all nitrogen-fixing strains  and a bidirectional hydrogenase (HoxFUYH) which is involved in both hydrogen synthesis and oxidation but seems to be unrelated to the nitrogen-fixing process . In Synechocystis PCC 6803, the bidirectional HoxEFUYH hydrogenase functions for H2 uptake or production . In V.limneticus sp. nov. we detected only this bidirectional hydrogenase (HoxEFUYH). This is surprising since almost all nitrogen-fixing cyanobacteria possess also the uptake hydrogenase (HupSL) except for Synechococcus BG 043511 . Hence, it appears that in some strains of N-fixing Synechococcus there is an absence of the uptake hydrogenase. The recycling of hydrogen by these type of enzymes generates an anoxic environment necessary for nitrogenase activity  and the H2 production could be assumed both by nitrogenases and bidirectional hydrogenases .
Regarding sulfur metabolism, we observed mechanisms for uptake and utilization of alkanesulfonates in V.limneticus sp. nov. Two alkanesulfonate monooxygenases and six sulfonate ABC transporters (TauABCD and ssuEADCB) were detected in the genome. These organosulfur compounds were also reported to be degraded by freshwater Actinobacteria  and Achromobacter or Rhodococcus strains . The utilization of these sulfur sources by our picocyanobacterium is ecologically relevant since sulfonates tend to accumulate in soils, rivers, groundwater  and marine sediments  and some of them like atmospheric methanesulfonates  or herbicides  are considered contaminants and hazardous compounds for the environment and very harmful for animals and humans. We also found two different clusters of the specific freshwater sulfate Cys transporter which was previously reported in freshwater Cyanobium and Synechococcus . A chitinolytic enzyme and key enzymes for the N-acetylglucosamine utilization (NAG core enzymes nagA, nagB and the NAG kinase nagK) were also detected in the genome, which suggests that V.limneticus sp. nov. may also be capable of chitin degradation. All these features enhance the ecological relevance of the novel V.limneticus sp. nov.
This study shows that Vulcanococcus limneticus sp. nov. possess a complete nitrogenase and nif operon, possibly acquired by HGT and located in a genomic island. However, in our experimental conditions, V.limneticus sp. nov. did not express the nifHDK genes. We cannot eliminate the possibility that in other conditions the nifHDK genes might still be expressed. Nevertheless, the presence of genes coding for different enzymes in the V.limneticus sp. nov. genome like alanine dehydrogenase or hydrogenase points to other pathways to incorporate ammonia, like direct ammonia assimilation from phycobilisome degradation, nitrate/nitrite ammonification or cyanate hydrolysis, which are energetically less expensive for the cell.
Description of Vulcanococcus limneticus sp. nov
Vulcanococcus (Vul.ca.no.coc’cus, L. masc. n. vulcanus volcan [referring to the volcanic region from which the organism was isolated]; N.L. masc. n. coccus of spherical shape); limneticus limnetic (lim.ne.ti’cus, L. masc. Adj. [referring to lacustrine origin of the organism]).
The isolation source was the freshwater volcanic Lake Albano located in central Italy. It is composed by aerobic gram-negative non-motile cells approximately 0.97 ± 0.21 μm long and 0.76 ± 0.12 μm wide (volume: 0.36 ± 0.19 μm3). It can form microcolonies of 10–20 cells. Vulcanococcus limneticus sp. nov. presents a G + C content of 68.35 mol%. The genome has a total size of 3,548,882 bp. The strain grows optimally in BG11 media for freshwater cyanobacteria at neutral pH and between 19 and 25 °C at low light (10–15 μmol photons m− 2 s− 1). The strain has a maximum absorbance at 573 nm typical of phycoerythrin and develops a type IIB intense pink-red pigmentation.
The strain used for this experiment was a phycoerythrin-rich (PE) picocyanobacteria . This picocyanobacterium has been isolated from a volcanic lake in central Italy (Lake Albano, Additional file 1: Table S1) using cycloheximide (with a final concentration of 3 mmol l− 1) to eliminate the picoeukaryotes. Then the culture was purified by sorting (InFlux V-GS flow cytometer, Becton Dickinson Inc.) one single cell each into wells of 96-well plates containing 100 μl of BG11 substrate and kept in a thermostat at 18–20 °C at low light (10–15 μmol photons m− 2 s− 1) . In this way we obtained a monoclonal culture (derived from one single mother cell) not axenic (with associated microbiome, ). This strain has been used in different experiments [24, 55] and was named Synechococcus LL based on 16S rDNA. The cells with phycoerythrin are well recognized by the flow cytometer, forming a defined cloud in the cytograms with signal collected in orange and red channels . Recently it has been sequenced for the entire genome (accession number NQLA01000000, Sanchez-Baracaldo et al., submitted) and following the recent Cyanobacteria classification scheme  based on GGDH, ANI and AAI delineation parameters (Additional file 3: Table S2) it belongs to a new species from a novel genus and it has been renamed as Vulcanococcus limneticus sp. nov.
DNA was extracted using AXG (Machery-Nagel, Düren, Germany) gravity flow columns as per the manufacturer’s protocol. Library prep was performed with the Illumina TruSeq Nano DNA Library Preparation Kit (Illumina, San Diego, CA) and sequenced on an Illumina Hi-Seq 2500. Reads were trimmed using Trimmomatic v0.32  before being assembled using SPAdes v3.5.0 . Contaminating sequences were removed by identifying cyanobacterial contigs with a database of core cyanobacterial genes  using tBLASTn v2.2.30+ (e-value threshold of 1e− 10) and visualising using Bandage v0.07  as previously described . The final assembly consisted of 160 contigs and is deposited on NCBI GenBank under the accession number NQLA01000000.
A reference protein-concatenate-based tree with a total of 259 universal markers based on PhyloPlAn tool  was created, using a total of 111 Synechococcus and Cyanobium genomes originating from multiple habitats. Nine Prochlorococcus marinus were also used to complete the phylogeny.
Single gene trees
A NifHDK protein concatamer tree was constructed with NifHDK concatamers from a wide range of bacteria as previously described [35,36,37,38]. Sequences were aligned using MAFFT  and trees were constructed with FastTree2, using JTT + CAT model, a gamma approximation and 100 bootstraps.
Comparison of the nitrogenase nif operon among different Cyanobacteria
The structure and similarity of the nitrogenase subunits for the different compared cyanobacteria, including the novel planktonic Vulcanococcus limneticus sp. nov., was assessed by tBLASTX  with > 50% similarity hits and 50 bp of alignment lengths.
Genome annotation and metabolic pathways
Metagenomic fragment recruitment of Vulcanococcus limneticus sp. nov. on Amazon lake datasets
We performed recruitment plots following methods from other publications . We used Lake Ananá and Mancapuru Great lake metagenomic datasets to evaluate the presence of V.limneticus sp. nov. and its low covered genomic islands in the tested metagenomes .
We carried out a laboratory experiment with Vulcanococcus limneticus sp. nov., in conditions in which it grows very well, by simply using two treatments, with (+N) and without (-N) nitrogen in the culture media, to measure nifHDK transcripts under nitrogen limitation conditions. In this way, we were able to use a non-axenic culture to recognize the activation of the nifHDK gene for the nitrogenase activity specific for V.limneticus sp. nov. The culture media was specific for cyanobacteria with nitrogen present as NaNO3 at a final concentration of 8 mmol L− 1 of N in the medium and N: P of 20 . We added 1 ml of a dense culture (inoculum) to 50 ml of each medium with (+N) and without (-N) nitrogen to reach starting abundances of V.limneticus sp. nov. around 8 × 105 cells ml− 1. The volume of the inoculum was low, so that the nitrogen added in the –N treatment was considered negligible. Each treatment was performed in triplicate, in semi-continuous cultures, kept in the same condition of maintenance in a culture room at 20 °C, with a 12 h light: 12 h dark diel-cycle using cool white fluorescent tubes at an intensity of 20 μmol photons m− 2 s− 1. Each day at the end of the light cycle and at the beginning of the dark cycle (Additional file 4: Fig. S1) 0.5 ml were taken from each treatment and fixed for counting (0.2 μm filtered formaldehyde at 1% final concentration). We followed the growth of the culture calculating the daily growth rate and when the culture was in exponential phase (on ninth day), we sampled for the RNA analysis at the beginning of the dark cycle as previously suggested . The culture flasks were incubated at the same experimental conditions for one month to control the state of the culture on a longer time-span.
Counting was performed on a flow cytometer Accuri C6 (Becton Dickinson Inc., New Jersey, US), equipped with a 50 mW laser emitting at a fixed excitation wavelength of 488 nm. The instrument provides light scattering signals (forward and side light scatter named FSC and SSC, respectively), green fluorescence (FL1 channel = 533/30 nm), orange fluorescence (FL2 channel = 585/40 nm) and red fluorescence (FL3 channel > 670 nm and FL4 channel 675/25). For counting we used FL2-H vs FL3-H which better singled out the phycoerythrin-rich V.limneticus sp. nov. and allowed optimal gating design.
White polycarbonate filters (Poretics, 0.2 μm pore size) were used for microscopic evaluation of the cells at the beginning of the experiment, at the sampling for RNA analyses and at the end of the experiment. We used a Zeiss Axioplan microscope equipped with an HBO 100 W lamp, a Neofluar 100 x objective 1.25 x additional magnification and filter sets for blue (BP450–490, FT510, LP520) and green light excitation (LP510–560, FT580, LP590).
RNA extraction and transcription analysis
The RNA extraction was carried out from the broth cultures of the strain of V.limneticus sp. nov. cultivated in the presence and absence of nitrogen. Aliquots of the all replicates were analyzed for the abundance of V.limneticus sp. nov. by flow cytometry as described above. A volume of 20 ml of culture correspondent to 108 cells ml− 1 was centrifuged for 5 min at 13000 g and the pellets were processed for the RNA extraction using a commercial kit following the manufacturer’s instructions (RNeasy Protect Bacteria Mini Kit, Qiagen) with some modifications. The chemical and mechanical lysis were conducted together (4 cycles of 6000 rpm for 30s, using the Precellys 24 homogenizer, Bertin technology). Moreover, a DNase (RNase-Free DNase, Qiagen) treatment was carried out before the RNA purification. The RNA concentration was evaluated by fluorometric approach using Qubit assay following the manufacturer’s instructions (Qubit RNA HS Assay Kit, Invitrogen Life Technology). Comparable amounts of RNA (around 50 ng) of each sample were retro-transcribed following the manufacturer’s protocol (QuantiTect Reverse Transcription, Qiagen). NORT for each sample was used as non-template control for the estimation of genomic DNA amplification signal. All the samples of cDNA and NORT were tested for the presence/abundance of the nitrogenase genes (nifHDK) and of 16S rDNA of V.limneticus sp. nov. by real time PCR. The primers for all the tested genes were designed using the genome sequence of V.limneticus sp. nov. as template using NetPrimer software (http://www.premierbiosoft.com/netprimer/index.html), the specificity of each primer was verified by blasting all the primer sequences against that of the V.limneticus sp. nov. full genome. The primer sequences are reported in supplementary material (Additional file 7: Table S4). All real time PCR assays were carried out using RT-thermocycler CFX Connect (Bio-Rad), following the same programs described elsewhere , except for changing the annealing temperatures (Additional file 7: Table S4). In all the real time PCR assays DNA extracted from V.limneticus sp. nov. was used as positive control. The specificity of the reaction for each sample was ensured by the melting profile analysis using the PRECISION MELT ANALYSIS Software 1.2 built in CFX MANAGER Software 3.1 (Biorad) and by the electrophoresis gel run. The abundance of each gene for all the samples was expressed as CT value.
Average Amino acid Identity
Average Nucleotide Identity
- fdx :
Forward light scatter
Genome-to-Genome DNA Hybridization
Horizontal Gene Transfer
Hydrogenase Uptake activity
- modAB :
- nif :
NOn-Reverse Transcribed RNA
Side light scatter
Monooxygenases sulfonate ABC transporter
Alakanesulfonate ABC transporter
Xenobiotic Response Element
Stal LJ. Nitrogen fixation in cyanobacteria. In: Encyclopedia of life sciences (ELS). Chicester: John Wiley & Sons, Ltd; 2008. https://doi.org/10.1002/9780470015902.a0021159.
Brauer VS, Stomp M, Bouvier T, Fouilland E, Leboulanger C, Confurius-Guns V, et al. Competition and facilitation between the marine nitrogen-fixing cyanobacterium Cyanothece and its associated bacterial community. Front Microbiol. 2015;5:795.
Carpenter EJ. Nitrogen fixation by marine Oscillatoria (Trichodesmium) in the world's oceans. In: Carpenter EJ, Capone DG, editors. Nitrogen in the marine environment. New York: Academic Press, Inc.; 1983. p. 65–103.
Zehr JP, Jenkins BD, Short SM, Steward GF. Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol. 2003;5:539–54.
Dos Santos PC, Fang Z, Mason SW, Setubal JC, Dixon R. Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Gen. 2012;13:162.
Welsh EA, Liberton M, Stöckel J, Loh T, Elvitigala T, Wang C, et al. The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle. P Natl Acad Sci USA. 2008;105:15094–9.
Esteves-Ferreira AA, Cavalcanti JHF, Vaz MGMV, Alvarenga LV, Nunes-Nesi A, Araújo WL. Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions. Genet Mol Biol. 2017;40:261–75.
Berman-Frank I, Lundgren P, Falkowski P. Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res Microbiol. 2003;154:157–64.
Pancrace C, Barny M-A, Ueoka R, Calteau A, Scalvenzi T, Pédron J, et al. Insights into the Planktothrix genus: genomic and metabolic comparison of benthic and planktic strains. Sci Rep. 2017;7:41181. https://doi.org/10.1038/srep41181.
Zehr JP, Waterbury JB, Turner PJ, Montoya JP, Omoregie E, Steward GF, et al. Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean. Nature. 2001;412:635–8.
Zehr JP. Nitrogen fixation by marine cyanobacteria. Trends Microbiol. 2011;19:162–73.
Agawin NS, Benavides M, Busquets A, Ferriol P, Stal LJ, Aristegui J. Dominance of unicellular cyanobacteria in the diazotrophic community in the Atlantic Ocean. Limnol Oceanogr. 2014;59:623–37.
Shi T, Falkowski PG. Genome evolution in cyanobacteria: the stable core and the variable shell. P Natl Acad Sci USA. 2008;105:2510–5.
Latysheva N, Junker VL, Palmer WJ, Codd GA, Barker D. The evolution of nitrogen fixation in cyanobacteria. Bioinformatics. 2012;28:603–6.
Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, et al. Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. P Natl Acad Sci USA. 2013;110:1053–8.
Walter JM, Coutinho FH, Dutilh BE, Swings J, Thompson FL, Thompson CC. Ecogenomics and taxonomy of cyanobacteria phylum. Front Microbiol. 2017;8:2132. https://doi.org/10.3389/fmicb.2017.02132.
Flombaum P, Gallegos JL, Gordillo RA, Rincón J, Zabala LL, Jiao N, et al. Present and future global distributions of the marine cyanobacteria Prochlorococcus and Synechococcus. P Natl Acad Sci USA. 2013;110:9824–9.
Bhaya D, Grossman AR, Steunou A-S, Khuri N, Cohan FM, Hamamura N, et al. Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses. ISME J. 2007;1:703–13.
Rippka R, Waterbury J. The synthesis of nitrogenase by non-heterocystous cyanobacteria. FEMS Microbiol Lett. 1977;2:83–6.
Gushchin I, Chervakov P, Kuzmichev P, Popov AN, Round E, Borshchevskiy V, et al. Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria. P Natl Acad Sci USA. 2013;110:12631–6.
Spiller H, Shanmugam K. Physiological conditions for nitrogen fixation in a unicellular marine cyanobacterium, Synechococcus sp. strain SF1. J Bacteriol. 1987;169:5379–84.
Huang T-C, Chow T-J. New type of N2-fixing unicellular cyanobacterium (blue-green alga). FEMS Microbiol Lett. 1986;36:109–10.
Mitsui A, Kumazawa S, Takahashi A, Ikemoto H, Cao S, Arai T. Strategy by which nitrogen-fixing unicellular cyanobacteria grow photoautotrophically. Nature. 1986;323:720–2.
Callieri C, Coci M, Corno G, Macek M, Modenutti B, Balseiro E, et al. Phylogenetic diversity of nonmarine picocyanobacteria. FEMS Microbiol Ecol. 2013;85:293–301.
Guimarães PI, Leão TF, de Melo AGC, Ramos RTJ, Silva A, Fiore MF, et al. Draft genome sequence of the picocyanobacterium Synechococcus sp. strain GFB01, isolated from a freshwater lagoon in the Brazilian Amazon. Genome Announc. 2015;3:e00876–15.
Cabello-Yeves PJ, Haro-Moreno JM, Martin-Cuadrado A-B, Ghai R, Picazo A, Camacho A, et al. Novel Synechococcus genomes reconstructed from freshwater reservoirs. Front Microbiol. 2017;8:1151.
Rocap G, Larimer FW, Lamerdin J, Malfatti S, Chain P, Ahlgren NA, et al. Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature. 2003;424:1042–7.
Scanlan DJ, Ostrowski M, Mazard S, Dufresne A, Garczarek L, Hess WR, et al. Ecological genomics of marine picocyanobacteria. Microbiol Mol Biol Rev. 2009;73:249–99.
Dufresne A, Ostrowski M, Scanlan DJ, Garczarek L, Mazard S, Palenik BP, et al. Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria. Genome Biol. 2008;9:R90.
Sánchez-Baracaldo P. Origin of marine planktonic cyanobacteria. Sci Rep. 2015;5:17418.
Segata N, Börnigen D, Morgan XC, Huttenhower C. PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nature Com. 2013;4:2304.
Larsson J, Celepli N, Ininbergs K, Dupont CL, Yooseph S, Bergman B, et al. Picocyanobacteria containing a novel pigment gene cluster dominate the brackish water Baltic Sea. ISME J. 2014;8:1892–903.
Böhme H. Regulation of nitrogen fixation in heterocyst-forming cyanobacteria. Trends Plant Sci. 1998;3:346–51.
Masepohl B, Schölisch K, Görlitz K, Kutzki C, Böhme H. The heterocyst-specific fdxH gene product of the cyanobacterium Anabaena sp. PCC 7120 is important but not essential for nitrogen fixation. Mol Gen Genet. 1997;253:770–6.
Boyd ES, Hamilton TL, Peters JW. An alternative path for the evolution of biological nitrogen fixation. Front Microbiol. 2011;2:205. https://doi.org/10.3389/fmicb.2011.00205.
McGlynn SE, Boyd ES, Peters JW, Orphan VJ. Classifying the metal dependence of uncharacterized nitrogenases. Front Microbiol. 2013;3:419.
Boyd ES, Costas AMG, Hamilton TL, Mus F, Peters JW. Evolution of molybdenum nitrogenase during the transition from anaerobic to aerobic metabolism. J Bacteriol. 2015;197:1690–9.
Boyd ES, Peters JW. New insights into the evolutionary history of biological nitrogen fixation. Front Microbiol. 2013;4:201. https://doi.org/10.3389/fmicb.2013.00201.
Bandyopadhyay A, Elvitigala T, Welsh E, Stöckel J, Liberton M, Min H, et al. Novel metabolic attributes of the genus Cyanothece, comprising a group of unicellular nitrogen-fixing cyanobacteria. MBio. 2011;2:e00214–1.
Bolhuis H, Severin I, Confurius-Guns V, Wollenzien UI, Stal LJ. Horizontal transfer of the nitrogen fixation gene cluster in the cyanobacterium Microcoleus chthonoplastes. ISME J. 2010;4:121.
Toyama D, Kishi LT, Santos-Júnior CD, Soares-Costa A, de Oliveira TCS, de Miranda FP, et al. Metagenomics analysis of microorganisms in freshwater lakes of the Amazon Basin. Genome Announc. 2016;4:e01440–16.
Collier JL, Grossman A. A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria. EMBO J. 1994;13:1039.
Lahmi R, Sendersky E, Perelman A, Hagemann M, Forchhammer K, Schwarz R. Alanine dehydrogenase activity is required for adequate progression of phycobilisome degradation during nitrogen starvation in Synechococcus elongatus PCC 7942. J Bacteriol. 2006;188:5258–65.
Kamennaya NA, Post AF. Characterization of cyanate metabolism in marine Synechococcus and Prochlorococcus spp. Appl Environ Microbiol. 2011;77:291–301.
Ludwig M, Schulz-Friedrich R. Appel J occurrence of hydrogenases in cyanobacteria and anoxygenic photosynthetic bacteria: implications for the phylogenetic origin of cyanobacterial and algal hydrogenases. J Molecul Evol. 2006;63:758–68.
Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wünschiers R, Lindblad P. Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol Mol Biol R. 2002;66(1):1–20.
Cournac L, Guedeney G, Peltier G, Vignais PM. Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp. strain PCC 6803 deficient in the type I NADPH-dehydrogenase complex. J Bacteriol. 2004;186:1737–46.
Bothe H, Schmitz O, Yates MG, Newton WE. Nitrogen fixation and hydrogen metabolism in cyanobacteria. Microbiol Mol Biol R. 2010;74:529–51.
Ghai R, Mizuno CM, Picazo A, Camacho A, Rodriguez-Valera F. Key roles for freshwater Actinobacteria revealed by deep metagenomic sequencing. Mol Ecol. 2014;23:6073–90.
Erdlenbruch BN, Kelly DP, Murrell CJ. Alkanesulfonate degradation by novel strains of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and Rhodococcus sp., and evidence for an ethanesulfonate monooxygenase in A. xylosoxidans strain AE4. Arch Microbiol. 2001;176:406–14.
Boyd R. Herbicides and herbicide degradates in shallow groundwater and the Cedar River near a municipal well field, cedar rapids. Iowa Sci Total Environ. 2000;248(2):241–53.
Vairavamurthy A, Zhou W, Eglinton T, Manowitz B. Sulfonates: a novel class of organic sulfur compounds in marine sediments. Geochim Cosmochim Ac. 1994;58:4681–7.
Kelly DP, Murrell JC. Microbial metabolism of methanesulfonic acid. Arch Microbiol. 1999;172:341–8.
Callieri C, Amalfitano S, Corno G, Di Cesare A, Bertoni R, Eckert E. The microbiome associated with two Synechococcus ribotypes at different levels of ecological interaction. J Phycol. 2017;53:1151–8.
Callieri C, Amalfitano S, Corno G, Bertoni R. Grazing-induced Synechococcus microcolony formation: experimental insights from two freshwater phylotypes. FEMS Microbiol Ecol. 2016;92 https://doi.org/10.1093/femsec/fiw154.
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.
Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–77.
Mulkidjanian AY, Koonin EV, Makarova KS, Mekhedov SL, Sorokin A, Wolf YI, et al. The cyanobacterial genome core and the origin of photosynthesis. P Natl Acad Sci USA. 2006;103:13126–31.
Wick RR, Schultz MB, Zobel J, Holt KE. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics. 2015;31:3350–2.
Chrismas NA, Barker G, Anesio AM, Sánchez-Baracaldo P. Genomic mechanisms for cold tolerance and production of exopolysaccharides in the Arctic cyanobacterium Phormidesmis priestleyi BC1401. BMC Genomics. 2016;17:533.
Katoh K, Misawa K, Ki K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66.
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402.
Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2016;45:D200–3.
Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, et al. The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res. 2013;42:D206–14.
Jüttner F. Mass cultivation of microalgae and phototrophic bacteria under sterile conditions. Process Biochem. 1982;17:2.
Steunou A-S, Jensen SI, Brecht E, Becraft ED, Bateson MM, Kilian O, et al. Regulation of nif gene expression and the energetics of N2 fixation over the diel cycle in a hot spring microbial mat. ISME J. 2008;2:364.
Di Cesare A, Eckert EM, Teruggi A, Fontaneto D, Bertoni R, Callieri C, et al. Constitutive presence of antibiotic resistance genes within the bacterial community of a large subalpine lake. Molecular Ecol. 2015;24:3888–900.
Thanks to Jane Coghill and Christy Waterfall at the Bristol Genomics Facility.
Funding support for the genome analysis came from a Royal Society Research grant and a Royal Society Dorothy Hodgkin Fellowship for PS-B. Lab and publication costs were covered by CIPAIS (International Commission for Protection of Italian-Swiss Waters). The funding bodies had no role in the analysis, data interpretation and manuscript writing.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on request. Genome sequence is deposited on NCBI GenBank under the accession number NQLA01000000.
Ethics approval and consent to participate
No field permission was required for the collection of samples in this study.
Consent for publication
The authors declare that they have no competing interests
Table S1. Main characteristics of Lake Albano (Central Italy) from where V.limneticus sp. nov. was isolated. A more detailed study on water chemistry is published in Ellwood et al. 2009. This volcanic lake has trace level oxygen concentration in deep water (below 30 m), increase of NH4-N concentration below 70 m and calcite precipitation events in spring. (PDF 182 kb)
Fig. S2. Growth experiment of V.limneticus sp. nov. kept in two culture conditions: with and without nitrogen in the culture medium. The dark periods are reported as grey area. The number of cells are reported as mean of 3 replicates ± standard deviation. (PDF 337 kb)
Table S2. ANI (Average Nucleotide Identity, %), AAI (Average Amino acid Identity, %) and GGDH (Genome-to-Genome DNA Hybridization, %, expressed as identities/HSP length) between Vulcanococcus limneticus sp. nov. and phylogenetically closest species. (PDF 88 kb)
Fig. S1. Metagenomic fragment recruitment of V.limneticus sp. nov. concatenated genome on Amazon lake metagenomic datasets. A) Recruitment plot on Lake Ananá. B) Recruitment plot on Mancapuru Great lake. LPS biosynthesis and nif operon genomic islands are highlighted in red. (PDF 1778 kb)
Table S3. Abundance of the genes tested by real time PCR expressed as threshold cycle value. (PDF 83 kb)
Fig. S3. Microphotography at epifluorescence microscopy of V.limneticus sp. nov. cultures in the two treatments with (+N) and whithout (-N) nitrogen. Glycogen granules are clearly visible in the N limited culture. (Zeiss Axioplan, 1250×, blue excitation). V.limneticus sp. nov. monoclonal culture was isolated from a volcanic freshwater mesotrophic lake in central Italy, Lake Albano. Dimension of cells: 0.97 ± 0.21 × 0.76 ± 0.12 μm, Volume: 0.36 ± 0.19 μm3. (PDF 644 kb)
Table S4. Primer pairs used to quantify 16S rDNA and nif genes of V.limneticus sp. nov. (PDF 163 kb)
About this article
Cite this article
Di Cesare, A., Cabello-Yeves, P.J., Chrismas, N.A.M. et al. Genome analysis of the freshwater planktonic Vulcanococcus limneticus sp. nov. reveals horizontal transfer of nitrogenase operon and alternative pathways of nitrogen utilization. BMC Genomics 19, 259 (2018). https://doi.org/10.1186/s12864-018-4648-3
- Vulcanococcus limneticus sp. nov.
- Nitrogenase genes
- Nitrogen fixation
- Genomic island
- Horizontal gene transfer (HGT)