Our metagenomic data analysis predicts genes in GB-CYN1 involved in the Calvin cycle, TCA cycle, and photosystems I and II . Sequence identities for these genes in GB-CYN1 and published Chroococcales genomes were high, ranging from 75 to 99% . The observation of photosystem II genes in GB-CYN1 indicates the metabolic potential for oxygenic photosynthesis, unlike UCYN-A which lacks photosystem II, RuBisCo and the TCA cycle. These findings confirm that GB-CYN1 resembles free-living Chroococcales of the clades B and C rather than the metabolically streamlined, endosymbiotic UCYN-A clade . Related Chroococcales species are known to produce copious quantities of exopolysaccharides , and EPS produced by Crocosphaera watsonii has been linked to the formation of transparent exopolymeric particles in pelagic environments . A cassette of genes required for EPS production has been identified by comparative genomic analysis of related Crocosphaera watsonii strains ; however, we did not recover homologs of these genes in our metagenomic sequence . We conclude that the absence of these sequences is most likely due to our incomplete sampling of the GB-CYN1 genome, though it could also indicate either an unknown pathway for EPS synthesis in GB-CYN1, or an alternate source of the green berry exopolymeric matrix . Consistent with the dynamic oxygen conditions within the green berries, plastic plant pot we found sequences in the metagenomic data that suggest the metabolic potential for aerobic and anaerobic respiration, and anaerobic fermentative pathways assigned to several different phyla of bacteria .
Abundant fixed nitrogen from diazotrophy and transient anoxia presents an ecological opportunity for dissimilatory nitrogen metabolism, a process that could cause concomitant nitrogen fixation and loss over rapid spatiotemporal scales. Indeed, previous studies of diazotrophic Nodularia spumigena aggregates measured significant rates of both nitrification and denitrification . We specifically investigated potential marker genes for dissimilatory nitrogen metabolism within the green berries. Metagenomic sequences homologous to the nitrite reductase gene nirK were found in six unassembled reads and on a corresponding 753 bp long contig . A single read homologous to nitrous oxide reductase nosZ was also found suggesting the presence of a denitrification pathway . The top database matches to these nirK and nosZ sequences belonged to marine phytoplankton epiphytes from the Alphaproteobacteria and Flavobacterales . In the case of the nirK metagenomic sequences, the best match was to Roseibium sp. TrichSKD4, an alphaproteobacterial species isolated from a nitrogen-fixing Trichodesmium aggregate in the Atlantic Ocean . Homologs to the napA periplasmic nitrate reductase and the nirB nitrite reductase were identified from six and five unassembled reads, respectively, and were most similar to database sequences from marine heterotrophs in the Alphaproteobacteria and Gammaproteobacteria . We did not detect sequences supporting the presence of nitrifying bacteria or archaea, though given our limited sequence depth, this could be a function of missing data.
Cyanobacterial nitrogen fixation in the global oceans is frequently aggregate-associated, as with Trichodesmium sp. colonies and rafts , filamentous heterocystous cyanobacterial colonies , or Crocosphaera watsonii associated with TEP . The green berries of the Sippewissett Salt Marsh are nitrogen-fixing macroscopic consortia of unicellular cyanobacteria , diatoms and heterotrophic bacteria. While nitrogen and carbon fixation mediated by the green berries is unlikely to play a major role in the overall marsh ecosystem due to their low abundance and patchy distribution , these consortia provide an interesting comparative system to investigate the dynamics nitrogen flux within diazotrophic cyanobacterial aggregates. More broadly, studies of coastal marine estuarine sediments have indicated complex dynamics and close spatial coupling of co-occurring nitrogen fixation and denitrification processes . In other oceanic diazotrophic cyanobacterial aggregates, similarly rapid respiratory rates create transient anoxic zones within the aggregate core , and create a heterogeneous microenvironment where both aerobic and anaerobic metabolisms co-exist. This parallel is not merely conceptual: the green berry heterotrophic bacteria were similar, both phylogenetically and in their metabolic marker genes, to those observed to colonize other marine phytoplankton aggregates. The recovery of denitrification marker genesequences in metagenome suggests that there might be heterotrophic denitrifiers in the green berries with the potential to exploit this suboxic niche. However, further studies including rate measurements and better genome reconstructions are needed to clarify the importance of denitrification in the berries. The presence of such a pathway for nitrogen loss in the green berries is speculative, given the fragmented metagenome and absence of activity measurements, but remains an interesting direction for future work.
The existence of closely coupled nitrogen cycling within diazotrophic cyanobacterial aggregates has been explored previously, though initial studies demonstrating the association of heterocystous cyanobacterial aggregates with key bacterial species and marker genes for nitrification and denitrification measured only low to negligible rates . However, more recent investigations of in vitro rates and in situ marker gene transcription indicates that denitrification within anoxic cyanobacterial aggregates could contribute significantly to nitrogen loss in hypoxic waters, where bulk oxygen concentrations would normally inhibit such activity . In the light of modeling studies showing tight spatiotemporal coupling of nitrogen fixation and denitrification processes in the global ocean , we postulate that this coupling may be occurring at the microscale within ephemeral, aggregate associated niches. The potential for such interactions emphasizes the need to examine bio-geochemical cycles from the microbial perspective in the spatially heterogeneous marine environment.Green berries were sampled in June and July of 2010 or July and August 2014 from a single intertidal pool formed in the Little Sippewissett Salt Marsh, Falmouth, MA United States . These aggregates, however, have also been observed at other locations throughout Little and Great Sippewissett marshes. Berries were collected from the sediment-water interface by sieving and were washed three times in 0.2 µm filter sterilized marsh water. Imaging of the berries was conducted using a Zeiss Axio IMAGER MZ epifluorescence microscope equipped with a color camera and a Zeiss LSM 710 spectral confocal scanning laser microscope .The MG-RAST 3.3 pipeline was used to classify unassembled metagenomic sequence reads as ribosomal RNA and protein coding sequences using the M5RNA and M5NR databases to provide a diversity description shown in Figure 2 . Metagenomic sequence data was mined using MG-RAST. Functions of interest were mined using the hierarchical function assignment and were assigned to GB-CYN1 when the best hits were to sequenced Chroococcales genomes. Marker genes were further investigated via profile HMM and BLASTX searches . PCR amplified SSU rRNA gene sequences were aligned to the SILVA 115 database using SINA and curated using ARB . 16S rRNA phylogenies were inferred using the GTRGAMMA rate approximation. For functional genes of interest that were found to have frame shift sequencing errors , the sequences were corrected using Framebot . The metagenomic nifH amino acid sequence was aligned to references sequences from the nifH database available from the Zehr research group using the ARB software package . ProtTest3 was used to select the fixed WAG model of amino acid evolution with an inverse gamma rate approximation for the nifH phylogeny, plastic planter pot though similar topologies were recovered using related models. Phylogenetic reconstruction for both 16S rRNA and nifH genes were conducted as follows: maximum likelihood phylogeny was constructed with RAxML 7.2.8 with 1000 rapid bootstrap inferences and Bayesian phylogeny with MrBayes 3.3 . For Bayesian tree inferences, MCMC was run with default parameters and convergence was assessed when the reported average standard deviation of split frequencies fell below 0.01. Hidden Markov model profiles of 40 phylogenetic marker genes for Bacteria and Archaea were used to search the green berry metagenomic peptide sequence database using the trusted cutoffs. For each of the 34 markers that have green berry hits, green berry peptide sequences were aligned with all the bacterial and archaeal references sequences by hmmalign in HMMER3 . A maximum likelihood tree was built by Fastree2 for each alignments, and the alignments and trees were examined. As a result, 33 green berry peptide sequences were selected for further analysis because they branched with Cyanobacteria with good alignments. These sequences were further filtered to include only those 29 phyeco gene families with a single copy, cyanobacterial green berry hit. Single-copied reference sequences from 126 Cyanobacteria genomes of the 29 markers and the green berry cyanobacterial metagenomic sequences were retrieved from the alignments built in the previous step and were concatenated into a large alignment. A maximum likelihood tree was built using PHYML3.0 with the LG substitution model .
Tree topology and branch lengths were optimized by the program and SH statistics was used for branch support estimation.Whole-aggregate in vivo acetylene reduction assays were conducted on two 30 mL serum bottles of five washed green berries each. Acetylene, generated by the hydration of calcium carbide in an evacuated 150 mL serum-vial, was added to 10% of the headspace in a 30 mL serum-vial containing the berries in 5 mL of anoxic 0.2 µm filtered in situ marsh water under an 90% N2-CO2 atmosphere . Experiments were incubated on a 14 h light, 10 h dark cycle with full spectrum illumination at 30◦C for 2 days. Acetylene and ethylene were quantified using a Varian 2400 series gas chromatograph with an H2 flame ionization detector, as described previously . Rates were calculated over the 2 days incubation period with the assumption that nitrogen fixation was restricted to the dark hours of incubation, as previously described for unicellular cyanobacteria .Surface mulches are widely used in the production of strawberries and certain high value vegetable crops. Polyethylene mulch is used on virtually all tomato and strawberry production in Florida and is also widely used in the production of other crops such as peppers, eggplant, and melons throughout much of the southern United States. Researchers at the University of Florida estimate that more than 100,000 acres of vegetable crops in that state currently use plastic mulches annually, making Florida the national leader in this production system . In California, the majority of strawberry and staked tomato production uses polyethylene mulch. Peppers, eggplant, and melons also use mulches in certain situations, especially when earliness is desired. Field management and research related to plastic mulches in these production regions is now quite developed. Potential benefits as well as drawbacks of polyethylene mulches for vegetable crop production are given in table 1. The use of inter crop cover crop residues as surface mulches is a more recent and far less widely used production practice. It has recently gained considerable interest in a number of commercial vegetable crop production regions in the United States. The use of organic mulches in smaller-scale gardening contexts has had a very long history. Potential advantages and disadvantages of this vegetable crop production technique are summarized in table 2. Reflective plastic and some cover crop mulches share similar features relative to crop production: insect and disease management, weed management, fertilizer availability, and water conservation. In order for production practices using either polyethylene or cover crop mulches to be successfully adopted in California, specific production goals must be carefully matched with specialized management know how and experience.Plastic mulches have been commonly used for commercial vegetable crop production for more than 30years. Excellent sources of information on specific characteristics and applications of plastic mulches are available on the World Wide Web, and several of these are provided in the references to this publication. Most plastic mulch materials are made of either high- or low-density polyethylene, ranging from 0.30 to 0.79 inches in thickness, are 5 to 6 feet wide, and are available on rolls 555 to 1,338 yards long, depending on the thickness of the mulch . Black, white, and clear plastic mulches are most commonly used in commercial production, with black being the dominant color used for vegetables. Black plastic mulch is typically used for spring-seeded crops because it increases soil temperatures about 5ºF at a depth of 2 inches and 3ºF at 4 inches , compared to those of bare soil . Recently, a mulch has been introduced that consists of a strip of black plastic 30 cm wide down the center that is flanked on either side by metalized reflective plastic. This plastic combines the advantages of black plastic over the seed row, to help heat the soil, with the reflective characteristics of metalized plastic for insect and disease management. Black mulches have also recently been shown to reduce weed growth.