Open Access

Permanent draft genome of strain ESFC-1: ecological genomics of a newly discovered lineage of filamentous diazotrophic cyanobacteria

  • R. Craig Everroad1, 2Email author,
  • Rhona K. Stuart3,
  • Brad M. Bebout1,
  • Angela M. Detweiler1, 2,
  • Jackson Z. Lee1, 2,
  • Dagmar Woebken1, 4,
  • Leslie Prufert-Bebout1 and
  • Jennifer Pett-Ridge3
Standards in Genomic Sciences201611:53

https://doi.org/10.1186/s40793-016-0174-6

Received: 10 December 2015

Accepted: 15 August 2016

Published: 24 August 2016

Abstract

The nonheterocystous filamentous cyanobacterium, strain ESFC-1, is a recently described member of the order Oscillatoriales within the Cyanobacteria. ESFC-1 has been shown to be a major diazotroph in the intertidal microbial mat system at Elkhorn Slough, CA, USA. Based on phylogenetic analyses of the 16S RNA gene, ESFC-1 appears to belong to a unique, genus-level divergence; the draft genome sequence of this strain has now been determined. Here we report features of this genome as they relate to the ecological functions and capabilities of strain ESFC-1. The 5,632,035 bp genome sequence encodes 4914 protein-coding genes and 92 RNA genes. One striking feature of this cyanobacterium is the apparent lack of either uptake or bi-directional hydrogenases typically expected within a diazotroph. Additionally, a large genomic island is found that contains numerous low GC-content genes and genes related to extracellular polysaccharide production and cell wall synthesis and maintenance.

Keywords

CyanobacteriaNitrogen fixationHydrogenaseIntertidal microbial mat

Introduction

Microbial mats played a key role in the evolution of the early Earth and today provide a model system for exploring relationships between evolution, ecology, and biogeochemical cycles. In many mats, nitrogen-fixing filamentous Cyanobacteria are often central components with important roles in carbon, nitrogen and sulfur cycling [1, 2]. Recently, a previously unknown lineage of filamentous nitrogen-fixing Cyanobacteria was described in intertidal microbial mats from Elkhorn Slough, Moss Landing, California [3]. The type strain of this organism, ESFC-1, lacks both heterocysts and an extracellular sheath and has been shown to be an important cyanobacterial diazotroph in the Elkhorn Slough system [3]. At Elkhorn Slough this strain is often a dominant cyanobacterial member of the community (along with Cyanobacteria closely related to Coleofasciculus chthonoplastes PCC 7420); the sequence abundance of ESFC-1 in 16S rRNA libraries based on DNA and cDNA has been observed to reach up to 5 % (based on pyrosequencing) and 33–36 % (based on clone libraries and pyrosequencing), respectively [3, 4]. Although it is not always dominant, ESFC-1 is highly active, based on nifH transcript abundance and rRNA transcript to rRNA gene ratios [3, 5]. Recent work has shown that ESFC-1 produces a considerable external carbon pool as an EPS; this EPS is managed by means of an active exoproteome, and provides a source of organic carbon for the cyanobacterium and other community members [6]. Previous phenetic analyses using full-length 16S rRNA gene sequences have indicated this organism shares only a moderate identity with other identified Cyanobacteria ; its best cultured BLAST hit is the marine Aphanocapsa sp. HBC6 at 93.6 % similarity [7, 8]. Given the importance of ESFC-1 in the Elkhorn Slough mat system and its evolutionarily divergent 16S rRNA, the genomic sequence was determined [3, 8]. Here we report a detailed description of the genome of ESFC-1 as it relates to the ecology of this important mat community organism.

Organism information

Classification and features

Strain ESFC-1 was isolated by L. Prufert-Bebout at NASA Ames Research Center in Moffett Field, California from the top 2 mm of intertidal microbial mat samples collected at Elkhorn Slough, California, USA. Fresh microbial mat was repeatedly streaked onto plates of a modified version of ASN artificial seawater medium, until a unialgal culture was obtained [3, 9]. Strain ESFC-1 is a motile, Gram negative, non-heterocystous filament (Fig. 1). Trichomes are cylindrical in shape and straight to slightly curved, with rounded to slightly conical ends. Individual cells are approximately 1.8 μm across, and cells are typically longer than wide, up to 3.5 μm in length, slightly longer than reported previously [3]. Constrictions between cells are shallow but clearly visible. Hormogonia and akinetes have not been observed. Heterocysts have not been observed in cells, even when actively fixing N2. Morphologically, ESFC-1 appears most similar to isolates of the form-genus Geitlerinema , but with a cell size more typical of the form-genus Leptolyngbya [10].
Fig. 1

a Photomicrograph (400×) showing the filament morphology and size of Cyanobacterium ESFC-1, b Epifluorescent image of an ESFC-1 biofilm. Image is a calculated maximum intensity projection of a 50 μm z-stack. Red is autofluorescent ESFC-1 trichomes. Cells were fixed with 10 % formaldehyde prior to imaging. c Scanning electron microscopy image of ESFC-1 trichomes. ESFC-1 samples were fixed with 10 % formaldehyde, rinsed with sterile water, spotted onto a silicon wafer, air-dried and coated with ~5 nm of gold. Imaged with an FEI Inspect F SEM (Hillsboro, OR). For all panels, scale bar represents 10 μm

General features of ESFC-1 and project information are presented in Tables 1 and 2. In previous similarity and phylogenetic analyses based on the 16S rRNA locus, strain ESFC-1 did not show a close similarity (<94 %) with any other cyanobacterial sequence, and its phylogenetic placement within the cyanobacterial radiation was ambiguous [3, 8]. A 31-marker gene phylogenomic analysis of the cyanobacterial radiation, including ESFC-1, is presented in Fig. 2. This analysis places ESFC-1 with strong support in a clade with two non-diazotrophic Spirulina strains, PCC 6313 and PCC 9445 [10].
Table 1

Classification and general features of strain ESFC-1 according to the MIGS recommendations [38]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [39]

  

Phylum Cyanobacteria

TAS [40]

  

Class Cyanobacteria

TAS [10]

  

Order Oscillatoriales

TAS [10]

  

Family Oscillatoriaceae

TAS [10]

  

Genus Unclassified

TAS [3]

  

Species Unclassified

TAS [3]

  

strain: ESFC-1

 
 

Gram stain

Negative

NAS

 

Cell shape

Cylindrical cells in filaments

TAS [3]

 

Motility

Motile

IDA

 

Sporulation

Not reported

NAS

 

Temperature range

Mesophilic

NAS

 

Optimum temperature

Unknown

NAS

 

pH range; Optimum

Unknown

NAS

 

Carbon source

Photoautotroph

TAS [3]

MIGS-6

Habitat

Marine/Intertidal

TAS [3]

MIGS-6.3

Salinity

Euryhaline; 3.5 % NaCl (w/v)

TAS [3]

MIGS-22

Oxygen requirement

Aerobic

NAS

MIGS-15

Biotic relationship

free-living

TAS [3]

MIGS-14

Pathogenicity

Non-pathogenic

NAS

MIGS-4

Geographic location

Elkhorn Slough, California, USA

TAS [3]

MIGS-5

Sample collection

October, 2009

TAS [3]

MIGS-4.1

Latitude

36°48′47″N

TAS [3]

MIGS-4.2

Longitude

121°47′5″W

TAS [3]

MIGS-4.4

Altitude

1.5 m

TAS [3]

aEvidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly ob-served for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [41]

Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

Improved High-Quality Draft

MIGS-28

Libraries used

Two Illumina PE libraries: 222 bp avg. insert, and 7791 bp avg. insert

MIGS-29

Sequencing platforms

Illumina

MIGS-31.2

Fold coverage

719×

MIGS-30

Assemblers

Velvet v. 1.1.05; ALLPATHS v. r38445; Phrap v. 4.24

MIGS-32

Gene calling method

Prodigal 2.5

 

Genbank ID

ARCP00000000

 

Genbank Date of Release

December 25, 2014

 

GOLD ID

Gi14129

 

BIOPROJECT

PRJNA165547

 

NCBI taxon ID

1128427

MIGS-13

Source Material Identifier

ESFC-1

 

Project relevance

Cyanobacterial ecology

Fig. 2

Maximum likelihood (ML) phylogenomic analysis of the cyanobacterial radiation based on a concatenated amino acid sequences for 31 conserved loci [35, 42] showing the phylogenomic affiliation of ESFC-1 with two species of Spirulina (PCC 6313 and PCC 9445; clade in red). Only the portion of the larger tree corresponding to lineage B2 (sensu [42]) is shown. The full 126-taxon ML tree was built using PHYML using the LG protein substitution matrix, and was rooted with Chloroflexus auranticus J-10, Rhodobacter sphaeroides 2.4.1, Heliobacterium modesticaldum Ice1, and Chlorobium tepidum TLS [4345]. ML bootstrap values for nodes >50 are shown; black boxes at the nodes denote bootstrap values of 100. Strain ESFC-1 and the nearest neighbors are highlighted in red

Genome sequencing information

Genome project history

Strain ESFC-1 was selected for sequencing because of its recent discovery as a major diazotroph in intertidal mat communities and its unique taxonomic position within the cyanobacteria. The genome project is deposited in the Genome On Line Database (GOLD Legacy ID Gi14129) and the complete genome sequence is deposited in GenBank (accession ARCP00000000). Sequencing, finishing and annotation were performed by the DOE-JGI. A summary of the project information is shown in Table 2.

Growth conditions and genomic DNA preparation

ESFC-1 was maintained in culture in liquid modified ASN at 25 °C on a 14:10 L:D cycle under cool fluorescent lamps at approximately 50 μmol photons · m-2 · s-1. High molecular weight genomic DNA was isolated based on the “JGI Bacterial DNA isolation CTAB” protocol from JGI [11], including an RNA digestion step according to the protocol. 50 μg of gDNA was provided to the JGI for sequencing.

Genome sequencing and assembly

The high-quality draft genome of strain ESFC-1 was generated by the DOE-JGI using the Illumina GAIIx platform [12]. An Illumina standard short-insert paired-end library with an average insert size of 222 bp +/− 50 bp generated 15,283,374 reads. An Illumina CLIP-PE long-insert paired-end library with an average insert size of 7791 +/− 660 bp generated 18,062,354 reads [13]. In total, 4099 Mbp of Ilumina data were generated.

The Illumina draft data was assembled with Allpaths, version r38445 [14], and contained 117 contigs in 14 scaffolds. The consensus was computationally shredded into 10 Kbp overlapping fake reads (shreds). The draft data was also assembled with Velvet, version 1.1.05 [15], and the consensus sequences were computationally shredded into 1.5 Kbp overlapping shreds. The Illumina draft data was reassembled with Velvet using the shreds from the first Velvet assembly to guide the reassembly. The consensus from this second Velvet assembly was shredded into 1.5 Kbp overlapping fake reads. Fake reads from the Allpaths and both Velvet assemblies were assembled using parallel phrap, version 4.24 (High Performance Software, LLC) with a subset of the Illumina CLIP-PE reads [16, 17]. Possible misassemblies were checked and manually corrected in Consed [18]. Gap closure was accomplished using repeat resolution software (Wei Gu, unpublished). The final assembly is based on 4099 Mbp of Illumina draft data, with an average genome coverage of 719×.

Genome annotation

The genome was annotated automatically with Prodigal 2.5 [19] by IMG [20], locally using the RAST server [21], and by GeneMarkS+ [22] by the NCBI annotation pipeline. Pathways of interest were mapped to the KEGG maps through both IMG and RAST.

Genome properties

The high quality draft genome of cyanobacterium ESFC-1 was resolved to 3 scaffolds consisting of 5,431,811, 135,349 and 64,875 bp, for a total of 5,632,035 bp. GC content was 46.47 %. The genome sequence is predicted to encode 5006 total genes, with 92 RNA genes, and 4914 protein-encoding genes. A majority (79.0 %) of genes were assigned putative functions, and the remainder were annotated as hypothetical proteins. The properties of the ESFC-1 genome, and the distribution of genes into COG functional groups are presented in Tables 3, 4, and Fig. 3. 16S rRNA gene sequence similarity to closely related cultured cyanobacteria, as determined by the phylogeny in Fig. 2, is summarized in Table 5 for the two 16S rRNA genes found in this genome.
Table 3

Genome statistics

Attribute

Value

% of total

Genome size (bp)

5,632,035

100.00

DNA coding (bp)

4,742,170

84.20

DNA G+C (bp)

2,617,048

46.47

DNA scaffolds

3

100.00

Total genes

5006

100.00

Protein coding genes

4914

98.16

RNA genes

92

1.84

Pseudo genes

146

2.92

Genes in internal clusters

880

17.58

Genes with function prediction

3505

70.02

Genes assigned to COGs

2625

52.44

Genes with Pfam domains

3742

74.75

Genes with signal peptides

253

5.05

Genes with transmembrane helices

1157

23.11

CRISPR repeats

19

0.25

Table 4

Number of genes associated with the general COG functional categories

Code

Value

% of total (2895)

Description

J

194

6.70

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

106

3.66

Transcription

L

94

3.25

Replication, recombination and repair

B

2

0.07

Chromatin structure and dynamics

D

27

0.93

Cell cycle control, cell division, chromosome partitioning

Y

0

0.00

Nuclear Structure

V

96

3.32

Defense mechanisms

T

230

7.94

Signal transduction mechanisms

M

223

7.70

Cell wall/membrane biogenesis

N

37

1.28

Cell motility

Z

0

0.00

Cytoskeleton

W

12

0.41

Extracellular Structures

U

33

1.14

Intracellular trafficking and secretion

O

145

5.01

Posttranslational modification, protein turnover, chaperones

C

139

4.8

Energy production and conversion

G

143

4.94

Carbohydrate transport and metabolism

E

201

6.94

Amino acid transport and metabolism

F

73

2.52

Nucleotide transport and metabolism

H

187

6.46

Coenzyme transport and metabolism

I

74

2.56

Lipid transport and metabolism

P

167

5.77

Inorganic ion transport and metabolism

Q

51

1.76

Secondary metabolites biosynthesis, transport and catabolism

R

398

13.75

General function prediction only

S

205

7.08

Function unknown

-

2381

47.56

Not in COGs

Fig. 3

Circularized representation of the three contigs of the genome of strain ESFC-1, with comparisons to Spirulina spp. PCC 9445 and PCC 6313, the closest relatives to ESFC-1 with genomes available. From center to outside: GC content, GC skew, genes on forward strand, genes on reverse strand. Red lines denote the location of the three contigs in this map. The identified genomic island begins at approximately 5050 kbp. Map was generated by BRIG [46]

Table 5

Near full-length 16S rRNA gene similarity matrix for ESFC-1, the two most-closely related cyanobacteria Spirulina spp. PCC 6313 and PCC 9445 (based on the phylogeny in Fig. 2), and the highest-scoring BLAST hit from the NCBI database (Aphanocapsa sp. HBC6, accession EU249123)

 

% Identities

Sequence

ESFC-1 01

ESFC-1 02

PCC 9445

PCC 6313 01

PCC 6313 02

EU249123

ESFC-1 01

1

0.984

0.899

0.905

0.906

0.923

ESFC-1 02

 

1

0.911

0.917

0.919

0.935

PCC 9445

  

1

0.915

0.916

0.913

PCC 6313 01

   

1

0.998

0.922

PCC 6313 02

    

1

0.923

EU249123

     

1

Insights from the genome sequence

ESFC-1 is a nitrogen-fixing cyanobacterium [3]; all structural genes for nitrogenase were detected (nifHDK operon; A3MYDRAFT_2398-2400), as were genes required for uptake of nitrate and reduction to ammonia (narB and nirA; A3MYDRAFT_3316 and A3MYDRAFT_3311, respectively). However, ESFC-1’s sister taxa, Spirulina PCC 6313 and PCC 9445, as determined by the phylogenomic analysis presented in Fig. 2, both lack the nif operon. Of the cyanobacterial taxa found in the phylogenomic tree, the highest scoring BLAST hit to the translated nifH sequence of ESFC-1 belongs to Halothece sp. PCC 7418 (second best score overall, 87 % amino acid identity, E-value 0.0). ESFC-1 has homologs required for assimilatory sulfur reduction, and a homolog for a sulfide:quinine oxidoreductase, suggesting it can utilize hydrogen sulfide as an electron donor to photosystem I; a useful trait in mat environments that may periodically become anoxic/sulfidic [23, 24].

ESFC-1 has a full complement of homologs for photosystems I and II, the cytochrome b6f complex, photosynthetic electron transport and ATP synthesis. Detected phycobilisome gene homologs indicate a phycocyanin-rich genotype, with phycocyanin, allophycocyanin core, and linker peptide homologs present. ESFC-1 appears to lack the ability to chromatically adapt; phycoerythrin and phycoerythrocyanin genes appear to be absent. A single set of phycocyanin homologs are present (cpcBA; A3MYDRAFT_2965-2964).

Complete sets of genes were detected for the Calvin-Benson cycle, the pentose phosphate pathway, Entner-Doudoroff pathway and glycolysis/gluconeogenesis. TCA cycle and the carbon dioxide concentrating mechanism genes were detected, with 18 hat/hatR homologs found. One locus, for malate dehydrogenase (EC 1.1.1.37) was not detected, however, a putative low-identity alternative protein homolog was found. Strain ESFC-1 contains the necessary gene homologs for most of the lactate and mixed acid fermentation pathways.

Extended insights

Cyanobacterium ESFC-1 appears to lack either a functional uptake or bi-directional hydrogenase. Neither the JGI nor RAST annotations detected these sequences. Extensive manual searches for the hox cluster genes (hoxEFUYH), encoding the bi-directional hydrogenase, and the hupSL genes encoding the uptake hydrogenase commonly found in N-fixing cyanobacteria, were unsuccessful. Similarly, the hydrogenase-maturation enzymes hypFCDE were not found, although the hypAB locus was detected (A3MYDRAFT_0781-0782). Comparative analyses of strain ESFC-1 with the two closely-related Spirulina strains revealed they both lack nif and hupL, but unlike ESFC-1, both possess hupS homologs and the hox operon. The hypFCDEAB homologs were found dispersed throughout their genomes; a comparative blastp analysis between the Spirulina PCC 6313 hox operon and best hits within strain ESFC-1 did not reveal any evidence of synteny for nearby loci.

The genome of ESFC-1 contains an approximately 56 kbp region of low GC content, with several putatively horizontally transferred ORFs (Fig. 3). Based on the IMG annotation, 51 ORFs were identified (A3MYDRAFT_4511 – A3MYDRAFT_4561), with a global GC content of 39.67 % and individual GC content for loci ranging from 29 to 47 %, compared with the global genome GC content of 46.47 %. At the phylum level and higher, IMG designated 112 ORFs as putatively horizontally transferred within the entire genome; of these 16, or 14.2 %, were found within this island (11 from Proteobacteria , and one each from Bacteroidetes , Chloroflexi , Deferribacteres , Firmicutes and Nitrospirae ). Additional blastp analysis against the non-redundant protein database (excluding environmental sequences) for these ORFs revealed 19 have best hits to non-cyanobacterial sequences. Several additional sequences in this island did have best hits to cyanobacterial sequences, but these cyanobacterial homologs appeared to be restricted to ESFC-1 and a few closely-related Cyanobacteria . The remaining high-scoring hits for these genes belonged to other bacterial phyla, suggesting that a gene transfer event for these loci into the Cyanobacteria occurred in a common ancestor shared by ESFC-1 and the other closely-related Cyanobacteria . In total, as many as 32 loci in the island region may have been horizontally transferred, either recently or into an ancestor of ESFC-1. Predicted gene functions for this region are primarily involved with lipopolysaccharide and outer membrane synthesis, including several methyltransferase-like and glycosyltransferase-like enzymes, such as homologs to the rfaB, rfaG, rfaS, loci and to the rfaL and rfbX loci related to O-antigen synthesis. Seven of these proteins have been detected via proteomics of an ESFC-1 culture, four of which were extracellular [6], suggesting genes in this island may play a role in extracellular polysaccharide and cell wall synthesis and maintenance. Many Cyanobacteria secrete exopolysaccharides with distinct structures and roles, both in protection from stress (ultraviolet radiation, osmotic and metals) and possibly for carbon storage [25]. This genomic island may provide an advantage to ESFC-1 in stress protection, as has been shown with other cyanobacterial genomic islands [26].

Conclusions

Despite representing a genus-level divergence within the Cyanobacteria , based on both 16S rRNA and phylogenomic analyses, the genome of ESFC-1 appears to belong to a typical filamentous cyanobacterium. However, the ESFC-1 genome is striking in its apparent lack of uptake or bi-directional hydrogenases expected within a diazotrophic cyanobacterium.

Although the uptake hydrogenase hupSL is found dispersed through the cyanobacterial radiation, to our knowledge, strain ESFC-1 is one of very few N-fixing cyanobacteria to lack this gene [27]. The uptake hydrogenase is generally considered an integral part of the energetically expensive process of N-fixation, allowing the cyanobacterium to recapture hydrogen produced by nitrogenase activity [28]. However, a deficient mutant of Anabaena sp. PCC 7120, lacking the large subunit of the uptake hydrogenase, hupL, demonstrated similar growth and N-fixation rates compared to the wildtype, but with enhanced hydrogen production under N-fixation conditions [29]. Given this, and the fact that ESFC-1 is known to be an active nitrogen-fixer in situ in the Elkhorn Slough intertidal mat community, it appears that some Cyanobacteria do not require a classical uptake hydrogenase, yet still perform this critical ecological role.

The bi-directional hydrogenase hox is also common within Cyanobacteria . It is thought to play roles in fermentation and potentially as an electron valve during photosynthesis to maintain proper redox conditions [3032]. However, as suggested by Tamagnini et al. [33], the physiological role of the bi-directional hydrogenase in Cyanobacteria is unsettled. A recent analysis of 36 cyanobacterial strains indicated that a bi-directional hydrogenase was necessary for hydrogen production via fermentation in these cyanobacteria [34]. Consistent with its apparent lack of a bi-directional hydrogenase, strain ESFC-1 has been shown to produce hydrogen under N-fixation, but not fermentation conditions under laboratory conditions (data not shown). One possible explanation is that strain ESFC-1 ferments under anoxic conditions via lactate or homolactate fermentation pathways, as found within the genome. Such fermentation is known from filamentous Cyanobacteria , and allows for maintenance of redox without concomitant production of hydrogen gas [30]. Both Spirulina spp. PCC 6313 and PCC 9445 possess homologues for the hox genes, so this absence in strain ESFC-1 is best explained by loss, consistent with the uneven distribution of the hup and hox genes in the cyanobacterial radiation [33].

Finally, since the ESFC-1 genome is not closed, the absent hox, hup and hyp genes are possibly in missing regions, or simply were not detected in the automated annotation process. However, extensive manual searches of the genome failed to find any putative hydrogenases. A search of the draft genome for the 107 marker genes commonly used to estimate completeness in metagenomic analyses found all 107 [35], suggesting the absence of these three gene groups is genuine.

Despite the apparent lack of a functional hydrogenase, strain ESFC-1 has been shown to be a dominant and active member of the Elkhorn Slough community. Further, it appears to be globally distributed. Although this distribution appears more limited compared to the cosmopolitan C. chthonoplastes, both nifH and 16S rRNA gene environmental sequences similar to ESFC-1 (>95 %) have been observed in the intertidal mats at Guerrero Negro, Mexico [36], and in lake sediments in Daqing, China (unpublished data, accession KJ176902). An isolate, Leptolynbya sp. LEGE 07176, from the intertidal zone in Portugal [37] may represent a second isolate of this lineage. As one of the only known N-fixing cyanobacteria natively lacking an uptake hydrogenase, this organism may be a suitable target for hydrogen production research. Future studies of ESFC-1 should experimentally confirm the lack of functioning hydrogenase proteins, and explore the nature and energetics of fermentation and N-fixation, and the ecological consequences for an organism that lacks these key enzymes.

Abbreviations

CLIP-PE: 

Cre-LoxP Inverse PCR Paired-End

EPS: 

Extracellular polymeric matrix

ESFC: 

Elkhorn Slough filamentous cyanobacterium

Declarations

Acknowledgements

Funding was provided by the US. DOE Genomic Science Program under contract SCW1039. Sequencing and support was provided by Community Sequencing Project #701 'Microbial Interactions in Extremophilic Mat Communities' at the DOE JGI. Work conducted by the U.S. DOE-JGI was supported by the Office of Science of the U.S. DOE Under Contract No. DE-AC02-05CH11231. Work at LLNL was performed under the auspices of the US DOE at Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. We thank Jeff Cann, Associate Wildlife Biologist, Central Region, California Department of Fish and Wildlife, for coordinating our access to the Moss Landing Wildlife Area.

Authors’ contributions

RCE drafted the manuscript, conceived the study, conducted the phylogenetics and light microscopy, and genome analysis. RKS performed the electron and fluorescence microscopy characterizations, genome analysis, and helped to draft the manuscript. LPB performed the cyanobacterial isolation, conceived the study, and participated in the study design. AMD and DW performed sample preparation and laboratory experiments, and participated in the study design. JZL participated in the study design and genome analysis. BMB and JPR conceived the study, and helped to draft the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Exobiology Branch, NASA Ames Research Center
(2)
Bay Area Environmental Research Institute
(3)
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory
(4)
Current address: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research Network “Chemistry meets Microbiology”, University of Vienna

References

  1. Bebout BM, Paerl HW, Crocker KM, Prufert LE. Diel interactions of oxygenic photosynthesis and N2 fixation (acetylene reduction) in a marine microbial mat community. Appl Eviron Microbiol. 1987;53:2353–62.Google Scholar
  2. Stal LJ. Physiological ecology of cyanobacteria in microbial mats and other communities. New Phytol. 1995;131:1–32.View ArticleGoogle Scholar
  3. Woebken D, Burow LC, Prufert-Bebout L, Bebout BM, Hoehler TM, Pett-Ridge J, et al. Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis. ISME J. 2012;6:1427–39.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Burow LC, Woebken D, Marshall IPG, Lindquist EA, Bebout BM, Prufert-Bebout L, et al. Anoxic carbon flux in photosynthetic microbial mats as revealed by metatranscriptomics. ISME J. 2013;7:817–29.View ArticlePubMedGoogle Scholar
  5. Burow LC, Woebken D, Bebout BM, McMurdie PJ, Singer SW, Pett-Ridge J, et al. Hydrogen production in photosynthetic microbial mats in the Elkhorn Slough estuary, Monterey Bay. ISME J. 2012;6:863–74.View ArticlePubMedGoogle Scholar
  6. Stuart RK, Mayali X, Lee JZ, Everroad RC, Hwang M, Bebout B, et al. Cyanobacterial reuse of extracellular organic carbon in microbial mats. ISME J. 2016;10:1240–51.View ArticlePubMedGoogle Scholar
  7. Foster JS, Green SJ, Ahrendt SR, Golubic S, Reid RP, Hetherington KL, et al. Molecular and morphological characterization of cyanobacterial diversity in the stromatolites of Highborne Cay, Bahamas. ISME J. 2009;3:573–87.View ArticleGoogle Scholar
  8. Everroad RC, Woebken D, Singer SW, Burow LC, Kyrpides N, Woyke T, et al. Draft genome sequence of an oscillatorian cyanobacterium, strain ESFC-1. Genome Announc. 2013;1:e00527–13.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Rippka R. Isolation and purification of cyanobacteria. Methods Enzymol. 1988;167:3–27.View ArticlePubMedGoogle Scholar
  10. Castenholz RW, Rippka R, Herdman M, Wilmotte M. Subsection III. (Formerly Oscillatoriales Elenkin 1934). In: Boone DR, Castenholz RW, editors. Bergey's manual of systematic bacteriology, vol. 539–562. 2nd ed. New York: Springer; 2001.Google Scholar
  11. Protocols - DOE Joint Genome Institute. http://jgi.doe.gov/user-program-info/pmo-overview/protocols-sample-preparation-information/. Accessed 25 May 2016.
  12. Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5:433–8.View ArticlePubMedGoogle Scholar
  13. Peng Z, Zhao Z, Nath N, Froula JL, Clum A, Zhang T, et al. Generation of long insert pairs using a Cre-LoxP inverse PCR approach. PLoS One. 2012;7:e29437.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Butler J, MacCallum I, Kleber M, Shlyakhter IA, Belmonte MK, Lander ES, et al. ALLPATHS: de novo assembly of wholegenome shotgun microreads. Genome Res. 2008;18:810–20.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Ewing B, Green P. Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res. 1998;8:186–94.View ArticlePubMedGoogle Scholar
  17. Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res. 1998;8:175–85.View ArticlePubMedGoogle Scholar
  18. Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998;8:195–202.View ArticlePubMedGoogle Scholar
  19. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:119.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Markowitz VM, Korzeniewski F, Palaniappan K, Szeto E, Werner G, Padki A, et al. The integrated microbial genomes (IMG) system. Nucleic Acids Res. 2006;34:344–8.View ArticleGoogle Scholar
  21. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST server: rapid annotations using subsystems technology. BMC Genomics. 2008;9:75.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Besemer J, Lomsadze A, Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 2001;29:2607–18.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Cohen Y, Jørgensen BB, Padan E, Shilo M. Sulfide-dependent anoxygenic photosynthesis in the cyanobacterium Oscillatoria limnetica. Nature. 1975;257:489–92.View ArticleGoogle Scholar
  24. Jørgensen BB, Revsbech NP, Blackburn TH, Cohen Y. Diurnal cycle of oxygen and sulfide microgradients and microbial photosynthesis in a cyanobacterial mat sediment. Appl Environ Microbiol. 1979;38:46–58.PubMedPubMed CentralGoogle Scholar
  25. Pereira S, Zille A, Micheletti E, Moradas-Ferreira P, De Philippis R, Tamagnini P. Complexity of cyanobacterial exopolysaccharides: composition, structures, inducing factors and putative genes involved in their biosynthesis and assembly. FEMS Microbiol Rev. 2009;33:917–41.View ArticlePubMedGoogle Scholar
  26. Stuart RK, Dupont CL, Johnson DA, Paulsen IT, Palenik B. Coastal strains of marine Synechococcus species exhibit increased tolerance to copper shock and a distinctive transcriptional response relative to those of open-ocean strains. Appl Environ Microbiol. 2009;75:5047–57.View ArticlePubMedPubMed CentralGoogle Scholar
  27. 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 Mol Evol. 2006;63:758–68.View ArticlePubMedGoogle Scholar
  28. Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wünschiers R, Lindblad P. Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol Mol Biol Rev. 2002;66:1–20.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Lindbald P, Christensson K, Lindberg P. Photoproduction of H2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: from laboratory to outdoor culture. Int J Hydrogen Energ. 2002;27:1271–81.View ArticleGoogle Scholar
  30. Stal LJ, Moezelaar R. Fermentation in cyanobacteria. FEMS Microbiol Rev. 1997;21:179–211.View ArticleGoogle Scholar
  31. Appel J, Schulz R. Hydrogen metabolism in organisms with oxygenic photosynthesis: hydrogenases as important regulatory devices for a proper redox poising? J Photoch Photobiol B. 1998;47:1–11.View ArticleGoogle Scholar
  32. Appel J, Phunpruch S, Steinmüller K, Schulz R. The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis. Arch Microbiol. 2000;173:333–8.View ArticlePubMedGoogle Scholar
  33. Tamagnini P, Leitão E, Oliveira P, Ferreira D, Pinto F, Harris DJ, et al. Cyanobacterial hydrogenases: diversity, regulation and applications. FEMS Microbiol Rev. 2007;31:692–720.View ArticlePubMedGoogle Scholar
  34. Kothari A, Potrafka R, Garcia-Pichel F. Diversity in hydrogen evolution from bidirectional hydrogenases in cyanobacteria from terrestrial, freshwater and marine intertidal environments. J Biotechnol. 2012;162:105–14.View ArticlePubMedGoogle Scholar
  35. Wu M, Eisen JA. A simple, fast, and accurate method of phylogenomic inference. Genome Biol. 2008;9:R151.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Woebken D, Burow LC, Behnam F, Mayali X, Schintlmeister A, Fleming ED, et al. Revisiting N2 fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach. ISME J. 2015;9:485–96.View ArticlePubMedGoogle Scholar
  37. Brito Â, Ramos V, Seabra R, Santos A, Santos CL, Lopo M, et al. Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: a polyphasic study. Syst Appl Microbiol. 2012;35:110–9.View ArticlePubMedGoogle Scholar
  38. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci. 1990;87(12):4576–9.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Castenholz RW. General characteristics of the cyanobacteria. In: Boone DR, Castenholz RW, editors. Bergey's manual of systematic bacteriology. 2nd ed. New York: Springer; 2001. p. 474–87.Google Scholar
  41. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.PubMedGoogle Scholar
  42. 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. Proc Natl Acad Sci U S A. 2013;110:1053–8.View ArticlePubMedGoogle Scholar
  43. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003;52:696–704.View ArticlePubMedGoogle Scholar
  44. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59:307–21.View ArticlePubMedGoogle Scholar
  45. Le SQ, Gascuel O. An improved general amino acid replacement matrix. Mol Biol Evol. 2008;25:1307–20.View ArticlePubMedGoogle Scholar
  46. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12:402.View ArticlePubMedPubMed CentralGoogle Scholar

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