In the format provided by the authors and unedited.

1

Supplementary Information 1

Main Article: Coordinated regulation of growth, activity and transcription in natural 2 populations of the unicellular nitrogen-fixing cyanobacterium Crocosphaera 3

Samuel T. Wilson1*, Frank O. Aylward1*, Francois Ribalet2, Benedetto Barone1, John R. Casey1, 4 Paige E. Connell3, John M. Eppley1, Sara Ferrón1, Jessica N. Fitzsimmons4, Christopher T. 5 Hayes5, Anna E. Romano1, Kendra A. Turk-Kubo6, Alice Vislova1, E. Virginia Armbrust2, David 6 A. Caron3, Matthew J. Church1†, Jonathan P. Zehr6, David M. Karl1#, Edward F. DeLong1# 7

1Daniel K. Inouye Center for Microbial Oceanography: Research and Education, Department of 8 Oceanography, University of Hawaii, Honolulu, HI 96822, USA 9

2School of Oceanography, University of Washington, Seattle, WA 98195, USA 10

3Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, 11 USA 12

4Department of Oceanography, Texas A&M University, College Station, TX 77843, USA 13

5School of Ocean Science and Technology, University of Southern Mississippi, Stennis Space 14 Center, MS 39529, USA 15

6Ocean Sciences Department, University of California, Santa Cruz, CA 95064, USA 16

†Current address: Flathead Lake Biological Station, University of Montana, Polson, MT 59860, 17 USA 18

*Samuel T. Wilson and Frank O. Aylward contributed equally to this work 19

#Corresponding authors: edelong@hawaii.edu; dkarl@hawaii.edu 20

Coordinated regulation of growth, activity andtranscription in natural populations of the unicellular

nitrogen-fixing cyanobacterium Crocosphaera

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONVOLUME: 2 | ARTICLE NUMBER: 17118

NATURE MICROBIOLOGY | DOI: 10.1038/nmicrobiol.2017.118 | www.nature.com/naturemicrobiology 1

2

Methods 21

Sampling. Seawater sampling was conducted using the ships underway system with the intake at 22

a depth of 9 m on the ship’s bow and a 24 x 12 L Niskin bottle rosette attached to a conductivity-23

temperature-depth (CTD) package (SBE 911Plus, SeaBird) with additional fluorescence, oxygen 24

(O2), and transmissometer sensors (Supplementary Figure 2). The fluorescence and O2 sensors 25

were calibrated using discrete measurements of chlorophyll a and phaeopigments1, and dissolved 26

O22, respectively. The mixed layer depth (MLD) was calculated based on an offset in seawater 27

density anomaly of 0.125 kg m-3

from the sea-surface (Supplementary Figure 2). Two periods of 28

intensive diel measurements were conducted during the cruise when CTD casts were conducted 29

every 4 h to a depth of 400 m and the entire 24 x 12 L Niskin bottle rosette was tripped at a depth 30

of 15 m corresponding to the depth of the drogue. Vertical profiles of water-column 31

biogeochemical properties were conducted at discrete depths of 5, 25, 45, 75, 100, 125, 150 and 32

175 m on 26, 30, 31 July, and 4 August 2017. To ensure consistency of measurements at Station 33

ALOHA, the sampling and analytical protocols for vertical profiles of pigments, nutrients, 34

particulates, and flow-cytometry enumerated phytoplankton populations (Prochlorococcus, 35

Synechococcus, picoeukaryotes) and heterotrophic bacteria were identical to those employed by 36

the HOT program (Supplementary Table S1) (http://hahana.soest.hawaii.edu/index.html). To 37

quantify dissolved iron (Fe) (<0.4 μm) concentrations, trace metal clean seawater using the 38

Moored In situ Trace Element Serial Sampler system an all-plastic module that opens and closes 39

an acid-cleaned high-density polyethylene (HDPE) bottle while underwater3. Seawater samples 40

for dissolved Fe analysis were filtered through acid-cleaned 0.4 μm polycarbonate track etched 41

filters into HDPE bottles and acidified at sea to pH 2 using ultrapure HCl. Approximately a year 42

after acidification, dissolved Fe concentrations were analyzed using an offline adaptation of the 43

SeaFAST pico metal pre-concentration system4, which extracts metals onto Nobias PA1 44

chelating resin at pH 6.5. Quantification was accomplished by isotope dilution after elution into 45

10% v/v Optima nitric acid. The eluent was analyzed on a Thermo Fisher Element XR ICP-MS 46

at the R. Ken Williams Radiogenic Laboratory, Texas A&M University. 47

Enumeration of Crocosphaera populations: The unicellular cyanobacteria were counted using 48

continual underway sampling as well as discrete sample analysis via microscopy and flow 49

3

cytometry. The continual underway sampling resolved the diel periodicity in cell abundance for 50

the small cells, the large cells were enumerated using the Attune Acoustic Focusing Flow 51

Cytometer, and microscopic measurements were used to verify cell sizes, as described below. 52

Underway measurements. Continuous measurements of Crocosphaera (along with 53

Prochlorococcus and Synechococcus) abundances and cell size were made using SeaFlow5. The 54

instrument was equipped with a 457 nm 300 mW laser (Melles Griot). Forward light scatter (a 55

proxy for cell size), red, and orange fluorescence were collected using a 457–50 bandpass filter, 56

692–40 band-pass filter, and 572–27 bandpass filter, respectively. Seawater was prefiltered 57

through a 100 µm stainless steel mesh (to eliminate large particles) prior to analysis. The flow 58

rate of the water stream was set at 15 mL min-1

through a 200 µm nozzle. A programmable 59

syringe pump (Cavro XP3000, Hamilton Company) continuously injected fluorescent 60

microspheres (1 µm, Polysciences) into the water stream as an internal standard. Data were 61

recorded to file in time intervals of 3 min and were analyzed using the R package Popcycle 62

version 0.2 (available on GitHub https://github.com/uwescience/popcycle). A sequential 63

bivariate manual gating scheme was used to identify the Crocosphaera population based on 64

forward light scatter, high orange (assumed to represent phycoerythrin-containing cells) and high 65

red fluorescence measurements. 66

67

Discrete: Discrete measurements of small and large-sized Crocosphaera abundances were made 68

using an Attune Acoustic Focusing Flow Cytometer (Applied Biosystems by Life Technologies) 69

with an excitation wavelength of 488 nm. Samples were fixed with microscopy-grade 70

paraformaldehyde (0.24% vol/vol final concentration), flash frozen in liquid nitrogen and stored 71

at -80°C until analysis on land. Pico- and nano-sized phytoplankton were counted directly after 72

thawing and the various groups discriminated based on their side scatter signals versus orange 73

(574 nm) fluorescence as well as their red (640 nm) versus orange fluorescence (Fig. 2a). To 74

ensure there was no carryover of cells between individual sample runs, ~200 µL of sample was 75

run through the instrument before starting data collection. We also verified that that the 76

population identified as ‘C1’ in Figure 2a is indeed the small cell Crocosphaera, by conducting 77

quantitative PCR (qPCR) analysis was conducted on fluorescence-activated cell sorted (FACS) 78

4

samples. qPCR assays of nifH for both UCYN-B (Crocosphera) and UCYN-C (Cyanthece-like) 79

were conducted on duplicate samples which each consisted of 500 sorted events. There was no 80

detection of UCYN-C and the UCYN-B nifH gene counts exceeded a value of 500 (ca. 1800 81

nifH gene copies per 500 cells) which is attributed to polyploidy. 82

Microscopy: The abundance and size of Crocosphaera were confirmed by fluorescence 83

microscopy (Supplementary Figure 3). Whole seawater was collected on 31 July at Station 48 84

from a depth of 15 m using a Niskin bottle, preserved in formaldehyde (1% final concentration), 85

and 100 mL was filtered onto a 25 mm, 0.2 μm, blackened polycarbonate and stained with 4’,6’-86

diamidino-2-phenylindole (DAPI; Sigma D9542). Crocosphaera cells were identified according 87

to their morphology and phycoerythrin autofluorescence when viewed by blue-light excitation 88

using epifluorescence microscopy. Crocosphaera cells (n=90) were then imaged on an Olympus 89

microscope equipped with a DP72 camera and cell diameters were determined using cellSens 90

Standard 1.11 software. 91

Abundance of diazotrophs and nifH gene sequencing. To characterize the N2 fixing 92

microorganisms, the nifH gene which encodes a subunit of the nitrogenase enzyme, was 93

quantified using quantitative PCR (qPCR). The groups of diazotrophs targeted included UCYN-94

A, Crocosphaera spp., Trichodesmium spp., and two types of heterocystous cyanobacteria that 95

form symbioses with diatoms (Supplementary Table S1). Discrete seawater samples (2 L) were 96

collected using the CTD-rosette, filtered using a peristaltic pump onto 10 µm polyester (GE 97

Osmotics, Minnetonka, MN) and 0.2 µm Supor (Cole Parmer, Vernon Hills, IL) filters in series, 98

frozen in liquid nitrogen, and stored at -80°C until processed. The DNA extraction was 99

conducted using published protocols6 and the qPCR analyses conducted as previously described

7. 100

Productivity. Productivity measurements included assimilation of 14

C-labeled bicarbonate 101

(NaH14

CO3) into particulate matter and quantification of the in situ ratio of oxygen to argon 102

(O2/Ar) using a membrane inlet mass spectrometer (MIMS). For the vertical profiles of in situ 103

14C incorporation, sampling protocols were identical for HOT cruises 104

(http://hahana.soest.hawaii.edu/index.html). Samples were collected at 5, 25, 45, 75, 100, and 105

125 m, fixed with NaH14

CO3, and incubated in situ from dawn to dusk in a free drifting array. 106

5

To quantify 14

C assimilation, seawater was filtered onto 25 mm diameter Whatman GF/F filters 107

and placed into scintillation vials. After acidifying with 1 ml of 2 M HCl and venting for 24 h to 108

remove inorganic 14

C, 10 ml of scintillation cocktail (UltimaGold LLT, PerkinElmer) was added 109

to each vial and the radioactivity counted on a Packard liquid scintillation counter 110

(TriCarb2770TR/LT) and quench corrected using internal protocols. Rates of 14

C incorporation 111

(14

C-PP) are reported per day and represent the net incorporation of carbon into particulate 112

matter during the daylight period. 113

For the in situ O2/Ar ratio measurements, discrete seawater samples were collected in triplicate at 114

a depth of 15 m using the CTD-rosette. The samples were collected in 12 mL Labco Exetainer® 115

screw cap vials, preserved with mercuric chloride, and analyzed on-board within 3–6 h using a 116

MIMS8. Briefly, the seawater sample is pumped at a constant flow rate (~2 mL min

-1) through 117

capillary stainless steel tubing and equilibrated to 23.00°C (±0.01°C) before passing through a 118

2.5 cm long tubular silicone membrane (Silastic®, DuPont), which has a vacuum on the outside 119

of the membrane. As the seawater sample flows through the membrane a fraction of the 120

dissolved gasses are transferred to the vacuum, where they pass through a liquid nitrogen trap (to 121

remove water vapor and carbon dioxide) before entering the ion source in the HiQuadTM 122

quadruple mass spectrometer (QMG 700). Reference measurements consisted of filtered (0.2 123

µm) surface seawater of known salinity and equilibrated with ambient air at 23.00°C (±0.01°C). 124

The concentrations of O2 and Ar in the standard were determined using the appropriate solubility 125

equations9,10

. 126

The deviation of O2/Ar from equilibrium in the mixed layer was calculated as: 127

(1)

where (O2/Ar)meas is the measured ratio, and (O2/Ar)sat is the ratio expected at saturation 128

equilibrium. 129

Net community production (NCP) was determined assuming that the mixed layer was in steady 130

state and that vertical and lateral mixing were negligible11

and using mean daily values of 131

(O2/Ar)8: 132

6

(2)

where kw is the weighted gas transfer velocity over the past 20 days (m d-1

)11

, is the 133

daily mean (O2/Ar), and [O2]eq is the O2 concentration at equilibrium for the mixed layer (mmol 134

m-3

). The NCP value calculated using this method averages over the residence time of O2 in the 135

mixed layer (~1 week during the study period) prior to the actual measurement. The gas transfer 136

velocity (kw) was calculated using the wind speed parameterization12

and wind speed at 10 m 137

above sea surface extracted at 24.5° N and 156.5° W using the Blended Sea Winds data 138

product13

, with a temporal resolution of 6 hours and a spatial resolution of 0.25 degrees. 139

Volumetric rates of NCP for the mixed layer were determined by dividing NCP, calculated using 140

equation (3), by the mixed layer depth. A photosynthetic quotient of 1.1 was used to convert 141

from O2 to C units14

. 142

Nitrogen fixation. Rates of N2 fixation were measured during the cruise using the 15

N2 143

assimilation technique. The 15

N-labeled gas was dissolved in filtered seawater prior to its 144

addition using filtered surface seawater collected at Station ALOHA15

and 15

N2 gas sourced from 145

Cambridge Isotope Laboratories. The quantities of nitrogen isotopes (i.e. N masses equivalent to 146

28, 29, and 30) were measured in each batch of 15

N2 enriched seawater using MIMS16

. The final 147

atom % enrichment in the seawater incubations averaged 5.72 ± 0.5 (SD). To conduct the rate 148

measurements in the field, 200 ml of 15

N2-enriched seawater was added to a 4 L polycarbonate 149

bottle which had been filled from a depth of 15 m collected with Niskin bottles attached to a 150

CTD rosette. Rates of N2 fixation were measured in triplicate every 4 h during 27-30 July and 151

31 July-3 August 2015. Samples were incubated for an average of 4 h using on-deck incubators 152

shaded to a light level equivalent of 15 m and maintained at near in situ temperatures which were 153

verified with underwater temperature data loggers (Hobo Pendant Data Logger; Onset Computer 154

Corporation). Upon termination of the incubation, the entire contents of the 4 L bottle were 155

filtered via a peristaltic pump onto a pre-combusted glass microfiber (Whatman 25 mm GF/F) 156

filter and stored at -20°C. On land, the filters were analyzed for the total mass of N and the 5N 157

composition analysis using an elemental analyzer-isotope ratio mass spectrometer (Carlo-Erba 158

EA NC2500 coupled with ThermoFinnigan Delta S) at the Stable Isotope Facility, University of 159

Hawaii. Internal standards consisting of dried plankton material were included in the analytical 160

7

run to evaluate instrument drift during analysis. To estimate Crocosphaera-specific rates of N2 161

fixation, we used the values from incubations conducted during the night period (1900-0600 hrs) 162

which at 7.3 ± 1.5 (SD) nmol N L-1

d-1

represent approximately two-thirds of the total rates of N2 163

fixation (10.9 ± 1.5 nmol N L-1

d-1

) (Table 1). 164

The night time rates of N2 fixation were attributed to Crocosphaera since it is the most abundant 165

diazotroph with an active nitrogenase during the night as indicated by the diel pattern of nifH 166

gene expression17

and observations of laboratory cultures18

. It is possible that noncyanobacteria 167

heterotrophic bacteria were also fixing N2 during the night period, however their estimated 168

contribution to N2 fixation during the dark is calculated to be <0.01%. This estimate is based on 169

measured nifH gene abundances of G24774A11, an uncultivated putative gamma proteobacteria, 170

of 8.8 ± 1.3 x 103 gene copies L

-1 and cell-specific rates of N2 fixation of 0.0013 fmol N cell

-1 h

-1 171

19. Therefore, even with active noncyanobacteria diazotrophic bacteria, their total contribution to 172

water-column N2 fixation is considered to be insignificant compared to Crocosphaera. 173

Biomass and growth rate estimates. To estimate the Crocosphaera biomass of small and large 174

cells, we computed carbon content cell-1

from biovolume20

using the equation: 175

log pg C cell-1

= log a + b x log V (μm3) (3) 176

whereby log a is the y-intercept (-0.583), b is the slope (0.860), and V is the biovolume (μm3) 177

calculated from the geometry of a sphere and cell diameter of 2.2 and 5.1 μm for the small and 178

large cells, respectively. Cell diameters were measured via microscopy and a threshold of 4 μm 179

was used to delineate the small and large cells (Supplementary Figure 3). The carbon content for 180

small and large Crocosphaera cells was therefore computed to be 1.2 and 10.1 pg C cell-1

, 181

respectively and applied to the cell counts as measured by the Attune cytometer (Fig. 2b). The 182

total biomass of Crocosphaera (i.e. small and large cells combined) was 0.04 ± 0.01 μmol C L-1

. 183

A growth rate for the small-sized Crocosphaera population was estimated from the increase in 184

cell abundances that occurred daily between 0600 to 1100 hrs, as measured by the SeaFlow. The 185

cell numbers used were the minimum cell abundance at 0600 hrs (± 30 mins) and the maximum 186

cell abundance at 1100 hrs (± 30 mins) during 25 July to 3 August. The derived growth rate was 187

0.6 ± 0.2 day-1

with a doubling time of 1.3 ± 0.4 days. Since the increase in cell abundances 188

8

reflects the net balance due to growth and mortality and we only take into account the cell 189

division that occurs between 0600–1100 hrs, these calculations of growth rate and doubling time 190

should be considered a conservative estimate. 191

Growth requirements and contribution to new production. To determine the relevance of 192

Crocosphaera metabolism, specifically nitrogen and carbon fixation, to community productivity 193

in the oligotrophic environment, two parameters were derived from the cell physiology and 194

water-column measurements: the nitrogen requirement of the Crocosphaera population and the 195

contribution of Crocosphaera to new production. 196

To calculate the cellular nitrogen requirement for the total Crocosphaera population (i.e. big and 197

small cells) we used the cell abundances, cell sizes, and estimated cell carbon content as 198

described in the previous ‘Biomass and growth rate estimates’ section to derive a standing stock 199

of Crocosphaera-specific carbon of 42.5 ± 6.4 nmol C L-1

(Fig. 2b). Using a Redfield molar 200

carbon:nitrogen ratio of 6.6, this was converted to Crocosphaera-specific nitrogen (Nstock) of 6.4 201

± 1.3 nmol N L-1

, which was translated into a daily nitrogen requirement (Nday) using equation 4: 202

Nday = Nstock x (e(0.58x1)

- 1) (4) 203

where 0.58 represents the growth rate (day-1

), as previously reported. Using the formula above a 204

Nday of 5.1 ± 3.4 nmol N L-1

d-1

is computed which is slightly lower than the Crocosphaera-205

specific rates of N2 fixation of 7.3 nmol L-1

d-1

(Table 1). From these comparisons of estimated 206

nitrogen requirement and measured rates of supply via N2 fixation, we surmise that nitrogenase 207

activity in Crocosphaera is closely regulated to meet the cellular N demand with little surplus 208

being released to the ambient environment. 209

The contribution of Crocosphaera to new production was assessed by converting the rate of 210

Crocosphaera-specific N2 fixation (7.3 nmol L-1

d-1

) to units of carbon, again using a Redfield 211

molar carbon:nitrogen ratio of 6.6, which yields 0.05 μmol C L-1

d-1

. These daily rates are 212

equivalent to 11% of NCP (0.45 ± 0.03 μmol C L-1

d-1

). The appropriateness of the Redfield 213

molar carbon:nitrogen ratio of 6.6 can be assessed by comparing with measured carbon:nitrogen 214

ratios of Crocosphaera strains in culture which varied between 6.0–8.5 (Mohr et al., 2010) and 215

9

5.0–10.0 (Dron et al., 2013). 216

Growth and grazing experiments. Growth and mortality (grazing) rates of Crocosphaera were 217

determined using a modified dilution method21,22

. Five-point dilution experiments were 218

conducted rather than 2-point experiments that are now sometimes employed23

. Five-point 219

curves provide a more sensitive indicator of non-linear relationships between dilution (grazer 220

abundance) and apparent phytoplankton growth rate. The method enabled the simultaneous 221

measurement of Crocosphaera growth (μ) and mortality (m) rates through the sequential dilution 222

of whole, unfiltered seawater (WSW) with 0.2 μm filtered seawater (FSW). Total phytoplankton 223

community growth and mortality rates were also determined from changes in chlorophyll a 224

concentrations (a proxy for total phytoplankton biomass) in each treatment. Four dilution 225

experiments were conducted on 26, 28, 31 July and 3 August with seawater collected at 2100 hrs 226

from a depth of 15 m using a Niskin sampling rosette and transferred into 23 L carboys, housed 227

in black bags to prevent photoshock of the phytoplankton assemblage. Filtered seawater was 228

prepared by filtering whole seawater through an acid-washed, DI rinsed, Pall 0.2 μm Acropak 229

1550 Capsule Filter with Supor Membrane. WSW was sequentially diluted with filtered seawater 230

to establish a five-point dilution series (100%, 80%, 60%, 40%, and 20% WSW) in acid-washed, 231

2.3 L, polycarbonate bottles. Nutrient stock (final incubation concentration of 2 μM NaNO3, 0.2 232

μM NH4Cl, 0.5 μM NaH2PO4·H2O, and 0.1 μM FeCl3·6H2O) was added to each bottle in the 233

dilution series to ensure consistent growth of all phytoplankton across all treatments. A 234

treatment of unenriched, 100% WSW bottles was also prepared to assess the impact of nutrient 235

addition on total phytoplankton and Crocosphaera growth rates. All treatments were prepared in 236

triplicate, with water gently transferred into the incubation bottles through acid-washed, silicone 237

tubing to reduce bubbling that harms delicate microzooplankton. Bottles were incubated for 24 h 238

using on-deck incubators shaded to a light level equivalent of 15 m and maintained at near in situ 239

temperatures. 240

To calculate population growth and mortality rates from the dilution experiments, total 241

phytoplankton rates were determined from changes in chlorophyll a concentrations, while rates 242

for the Crocosphaera assemblage were determined from cell abundances enumerated using flow 243

cytometry (FACSCalibur, Becton Dickinson, San Jose, CA), at the beginning (T0) and end (Tf) 244

10

of the incubation period (Supplementary Figure 4). Samples for Crocosphaera counts were 245

preserved with formalin (1% final concentration), flash-frozen in liquid nitrogen, and stored at -246

80°C. Triplicate flow cytometry samples were assessed from the T0 WSW and FSW and flow 247

cytometry samples were assessed from each bottle (treatments in triplicate) at Tf. Changes in 248

chlorophyll a (a proxy of total phytoplankton biomass) were determined from duplicate samples 249

collected from all bottles initially and at the end of the incubations. Aliquots were filtered onto 250

GF/F filters, which were extracted with 4 mL of 100% acetone at -20˚C overnight in the dark, 251

and measured using a Trilogy Laboratory Fluorometer (Turner Designs, San Jose, CA). Model I 252

linear regressions of chlorophyll a concentration and Crocophaera apparent growth rate (y-axis) 253

versus dilution factor (x-axis) were calculated to evaluate total phytoplankton and Crocosphaera 254

nutrient-enriched growth rates (μn; y-intercept of the regression) and mortality rates (m; slope of 255

the regression)21

. Intrinsic growth rates (μ0; growth rate of the phytoplankton in situ) were 256

calculated from growth in the unenriched, 100% WSW treatment and the mortality rate22

. 257

Intrinsic growth rates of Crocosphaera were highly variable (-0.16–0.99 day-1

), most likely due 258

to known effects of relatively low Crocosphaera cell abundances on the efficacy of the dilution 259

method and artifacts associated with bottle incubations. The grazing mortality rates ranged from 260

not significant (n.s.; 3 experiments) to 0.71 day-1

, the latter value was comparable to the doubling 261

times for Crocosphaera obtained using the underway flow cytometer (Table 1). 262

Sinking flux. The particulate nitrogen (PN) content of sinking material and its δ15

N isotopic 263

composition were determined from samples collected using two separate methods. The PN 264

content was measured in particles collected using individual collector traps situated at a depth of 265

150 m. Prior to deployment, the traps were filled with 0.5 µm filtered seawater solution 266

consisting of 50 g L-1

sodium chloride and 1% (vol/vol) formalin. Upon recovery, trap solutions 267

were pre-screened through 335 µm Nitex® mesh prior to filtration onto 25 mm diameter 268

combusted glass microfiber filters (Whatman GF/F). Post cruise, triplicate filters were processed 269

for PN analysis followed by quantification using an Exeter CE-440 CHN elemental analyzer 270

(Exeter Analytical, UK)24

. The δ15

N isotopic composition was measured in sinking particles 271

collected by a surface-tethered net trap deployed at 150 m25

. A sonar-triggered mechanism 272

closed the traps before retrieval, such that only particulate matter sinking to 150 m was collected. 273

11

On land, sample material was pre-screened through 335 µm Nitex® mesh prior to filtration onto 274

25 mm diameter combusted glass microfiber filters (Whatman GF/F). Six replicate filters were 275

analyzed for the total mass of N and the δ15

N composition analysis using an elemental analyzer-276

isotope ratio mass spectrometer (Carlo-Erba EA NC2500 coupled with ThermoFinnigan Delta S) 277

at the Stable Isotope Facility, University of Hawaii. 278

Genomics and transcriptomics 279

Sample Collection, Extractions, Library Preparation, and Sequencing 280

Seawater was collected at a depth of 15 m for the diel sampling and filtered with no pre-filtration 281

using a peristaltic pump onto 25 mm 0.2 μm Supor PES Membrane Disc filters (Pall, USA) 282

housed in Swinnex units. The filtration time ranged from 15–20 min and filters were placed in 283

RNALater (Ambion, Grand Island, NY) immediately afterwards and preserved at -80°C until 284

processing. DNA extractions were performed by thawing filters on ice, removing the RNALater, 285

and adding 400 μl of sucrose lysis buffer (final concentrations: 40 mM EDTA, 50 mM Tris (pH 286

8.3), and 0.75 M sucrose). Cell homogenization was performed using a Tissue Lyser (Qiagen, 287

Germantown, MD) programmed at 30 Hertz for two rounds lasting 1 min each. 100 μl of sucrose 288

lysis buffer containing 0.5 mg ml-1

lysozyme was added before incubating in a rotating hybrid 289

oven at 37°C for 30 min. Afterwards, 50 μl of sucrose lysis buffer containing Proteinase K (0.8 290

mg ml-1

) was added, followed by the addition of 50 μl of 10% SDS. Samples were incubated in 291

a rotating hybrid oven at 55°C for 2 hrs. DNA purification was robotically performed using 292

Chemagen MSM I instrument with the Saliva DNA CMG-1037 kit (Perkin Elmer, Waltham, 293

MA) and DNA quantification was determined using Picogreen dsDNA kit (Invitrogen, Waltham 294

MA). Subsequently, 250 ng of gDNA was sheared using Covaris M220 to a target insert size of 295

550 bp based on manufacture’s recommendation using Microtube-50 AFA fiber tubes. 296

Metagenomes were prepared for sequencing using Illumina’s TruSeq Nano LT library 297

preparation kit. RNA extractions were performed by removing RNALater followed by the 298

addition of 300 μl of Ambion denaturing solution directly to the filter then vortexed for 1 min. 299

Prior to purification, 750 μl of nuclease free water was added. Samples were robotically purified 300

and DNase treated using Chemagen MSM I instrument with the tissue RNA CMG-1212A kit 301

12

(Perkin Elmer, Waltham, MA). RNA quality was assessed using the Fragment Analyzer high 302

sensitivity reagents (Advanced Analytical Technologies, Inc.) and quantified using Ribogreen 303

(Invitrogen, Waltham MA). Metatranscriptomic libraries were prepared for sequencing with the 304

addition of 5–50 ng of Total RNA to the ScriptSeq cDNA V2 library preparation kit (Epicentre, 305

Chicago, IL). 306

Molecular standard mixtures used for quantitative transcriptomics were prepared as previously 307

described26

. Briefly, RNA standards were generated from DNA templates via T7 RNA 308

polymerase in vitro transcription (IVT) using the MEGAscript High Yield Transcription Kit 309

(Ambion). DNA templates were generated directly from the genome of Sulfolobus solfataricus 310

via PCR amplification and T7 promoter incorporation. Prior to RNA purification, 50 μl of each 311

standard group was added to the sample lysate targeting a final standard concentration of 312

approximately 1% to each sample based on expected total RNA yield, which for surface water 313

samples in the North Pacific Subtropical Gyre is typically 500 ng/L. Metagenomic and 314

metatranscriptomic samples were sequenced with an Illumina Nextseq500 system using V2 high 315

output 300 cycle reagent kit with PHIX control added for metagenomic (1%) and for 316

metatranscriptomic (5%) libraries27

. Both metagenomes and transcriptomes were multiplexed on 317

two runs each. The statistics for the paired-end reads generated in this manner are shown in 318

Dataset S1. 319

Bioinformatic Analyses: Identification of Crocosphaera Genes 320

Metagenomes from each time-point were assembled individually using Mira v. 4.9.5_228

, and 321

genes were subsequently predicted using Prodigal v. 2.629

(parameters –p, meta, and -c). Genes 322

were merged into an existing non-redundant gene catalog generated from metagenomes 323

sequenced from Station ALOHA (Mende et al., in review30

) using CD-HIT31

v. 4.6 (command 324

cd-hit-est-2d with parameters -aS 0.9, -c 0.95), and genes that were not incorporated were 325

clustered separately (command cd-hit-est parameters -aS 0.9, -c 0.95) and then added. Genes 326

and associated proteins from this non-redundant set were taxonomically classified through 327

comparison to the RefSeq 75 database32

using LAST v. 75633,34

(default parameters for 328

nucleotide comparisons, parameters “-b 1 -x 15 -y 7 -z 25 -e 80 -F 15” for amino acid 329

13

comparisons). All genes with a best hit (either nucleotide or protein) to a sequenced 330

Crocosphaera genome were manually curated to arrive at a set of 9,761 metagenome-derived 331

Crocosphaera genes that were used for subsequent transcript mapping. Functional annotations 332

for these genes were generated by comparing their amino acid sequences to the KEGG 333

database35

with LAST (parameters “-b 1 -x 15 -y 7 -z 25 -e 80 -F 15) and the EggNOG v. 4.1 334

database36

with HMMER337

. Annotations for the Crocosphaera genes analyzed in this study can 335

be found in Dataset S2. 336

Transcriptome Processing and Molecular Standard Normalization 337

Methods for transcriptome processing are similar to those previously described38

. Briefly, reads 338

were trimmed using Trimmomatic v. 0.27 (parameters: ILLUMINACLIP::2:40:15)39

, end-joined 339

using PandaSeq v. 2.4 (parameters: -F -6 -t 0.32, quality cutoff of 0.32)40

, and quality-filtered 340

using sickle v. 1.33 (length threshold set to 50)41

. Reads mapping to rRNA were then removed 341

using sortmerna v. 2.142

to arrive at the final set of non-rRNA reads. These reads were mapped 342

to the non-redundant set of Crocosphaera genes identified in the metagenomic data using LAST, 343

and a 95% ID cutoff was used to ensure high-quality mapping (the number of reads mapping to 344

genes used in this study is presented in Supplementary Figure 5). 345

To quantify recovery of the molecular standard spike-ins, non-rRNA reads were also mapped to 346

the standards using LAST. Normalization coefficients were calculated using previously 347

established methods26

. Four standards with zero reads mapping in at least one of the time-points 348

were not considered further given their low abundance. Moreover, five standards with 349

consistently high or low normalization coefficients were also removed to arrive at a set of five 350

standards that provided consistent results within each time-point (Standards S3, S5, S6, S10, and 351

S11). Plots of copies added versus copies recovered for these five standards are shown in 352

Supplementary Figure 6, and normalization coefficients and depth of sequencing estimates for all 353

standards are provided in Dataset S3. The normalization coefficients can also be viewed as 354

thresholds of detection, since one transcript mapping at a given timepoint would be calculated to 355

have a concentration equivalent to the normalization coefficient for that sample. It should be 356

noted that values of zero calculated here (due to zero transcriptomic reads mapping) should be 357

14

interpreted as values below the threshold of detection and not evidence for complete absence 358

from the sample. For each time-point the average normalization coefficient for these five 359

standards was multiplied by the reads mapped to each transcript in that sample to derive 360

estimates of transcripts per liter. This normalized count table was used for subsequent 361

bioinformatic analyses. 362

Identification of Transcripts Exhibiting Diel Oscillations 363

Analysis of temporal transcriptional patterns was restricted to a set of 1,978 Crocosphaera genes 364

that had on average ≥ 2 reads mapping per time-point in the transcriptomes to mitigate spurious 365

results from genes with low abundance. Temporal oscillations in the standard-normalized 366

abundances of these genes in the transcriptomes (heretofore referred to as transcripts) were 367

analyzed using Rhythmicity Analysis Incorporating Non-parametric Methods (RAIN)43

. This 368

non-parametric method allows for detection of transcripts exhibiting waveforms with 24-hr 369

periodicity without the need for fitting to a specific oscillating pattern, for example a sine wave. 370

Because there is no a priori reason to believe that diel transcript oscillations will all follow a 371

defined waveform, this approach allows for broad detection of transcripts with diel cycles. P-372

values resulting from this analysis were corrected for multiple testing using the false-discovery 373

rate method44

, and transcripts with corrected p-values ≤ 0.05 were considered significantly diel. 374

The estimate of total transcripts exhibiting diel oscillations derived from this approach can be 375

viewed as a lower bound, as it is likely that a robust diel signal could not be identified from some 376

low-abundance transcripts. Details regarding the annotation and diel oscillation tests for these 377

transcripts can be found in Dataset S2. 378

Network Analysis 379

Standard-normalized counts were analyzed using the package WGCNA in the statistical 380

programming language R using methods described previously45,46

. Briefly, a soft-threshold of 7 381

was chosen based on a scale-free network topology test, and transcriptional modules were 382

identified using the “blockwiseModules” command (minModuleSize = 30, mergeCutHeight = 383

0.25). Topological overlap between transcripts was calculated using the 384

TOMsimilarityFromExpr command, and module eigengenes, or first principle components, were 385

15

detected using the “moduleEigengenes” command47

. Networks were visualized with the R 386

package igraph48

. 387

16

Supplementary Information Figures and Table 388

Supplementary Table S1. Near-surface (0–50 m) nutrient concentrations and abundances of 389 key microorganisms. Values are reported as mean ± SD with n referring to the number of 390

vertical profiles that were conducted. For two parameters (dissolved iron and Crocosphaera 391 abundance for the large cells), in the absence of vertical profiles, we report the mean ± SD of 392 concentrations at a depth of 15 m. In the main document, the total biomass of the Crocosphaera 393 population (calculated as described in the ‘Supplementary Information) is compared with the 394 biomass of Prochlorococcus and Synechococcus, which was estimated using the cell abundances 395

listed in the Table below and a carbon content of 0.04 and 0.24, respectively49

. 396 397 Parameter Mean values 398 Nutrient concentrations (nmol l

-1) 399

nitrate + nitrite 8 ± 4 (n=5) 400 Phosphorus 57 ± 15 (n=5) 401 Silicate 1034 ± 92 (n=5) 402

Dissolved iron (<0.4 μm) 0.4 ± 0.1 (n=17) 403 404

Flow cytometry enumerated (cells l-1

) 405 Prochlorococcus 1.6 ± 0.6 x 10

8 (n=3)

406 Heterotrophic bacteria 5.1 ± 2.0 x 10

8 (n=3) 407

Synechococcus 1.1 ± 0.7 x 106

(n=3)

408 Picoeukaryotes 0.4 ± 0.3 x 10

6 (n=3) 409

Crocosphaera (small cells) 0.5 ± 0.2 x 106

(n=3) 410 Crocosphaera (large cells) 0.04 ± 0.02 x 10

6 (n=15) 411

412

nifH enumerated diazotrophs (gene copies l-1

) 413

UCYNA 8.7 ± 0.1 x 103

(n=1) 414 Crocosphaera 1.9 ± 0.4 x 10

6 (n=1) 415

Trichodesmium 3.0 ± 0.1 x 105

(n=1) 416

Heterocystous cyanobacteria 9.0 ± 0.1 x 105

(n=1) 417 Heterotrophic bacteria (γ-G24774A11) 8.8 ± 1.3 x 10

3 (n=1) 418

419

420

421

17

422

423

Supplementary Figure S1. Chart showing the sampling locations for selected measurements 424

along the cruise track during 25 July to 3 August 2015 when Lagrangian observations were 425

conducted. 426

18

427

Supplementary Figure S2. Upper water-column properties determined from vertical CTD 428

profiles every 4 h during July 25 to August 3 2015 (n=63) showing (a) temperature, (b) salinity, 429

(c) oxygen, and (d) chl a + phaeopigments. For each parameter the sampling frequency was 24 430

Hz and the vertical depth resolution is 2 m. The solid white line represents the depth of the 431

mixed layer based on a density offset of 0.125. 432

19

433

Supplementary Figure S3. Abundance and size of the Crocosphaera populations. (a) 434

Abundance of small Crocosphaera cells counted with the Attune cytometer (grey circles) 435

(technical replicates, n=3) shown against the continual measurements (solid grey line) from Fig. 436

2 in the main document, (b) Abundance of large Crocosphaera cells counted with the Attune 437

cytometer (technical replicates, n=3) (c) Microscopy measurements of cell diameter sampled on 438

31 July 2015 (n=90). 439

20

440

21

441

Supplementary Figure S4. Results of the dilution experiment conducted 28 July 2015. Data 442

points indicate apparent growth rates obtained from the dilution series (1.0 is undiluted seawater, 443

0.2 is 20% undiluted seawater and 80% diluent). Black symbols are data from the nutrient-444

enriched dilution series, red symbols are data from the unenriched treatment (which indicate net 445

growth rates at ambient nutrient concentrations). Intercepts indicate intrinsic growth rates, slopes 446

indicate mortality (grazing) rates (both in units of d-1

). (a) Results for total chlorophyll a (i.e. 447

whole phytoplankton community). (b) Results for Crocosphaera. Vertical lines are standard 448

deviations among the triplicate samples at each dilution (standard deviations among triplicate 449

samples for the chlorophyll analyses were generally smaller than the size of the symbol). 450

22

451

Supplementary Figure S5. Histogram of transcript abundances before normalization (i.e., hit 452

counts) for the 1,978 transcripts identified throughout the cruise. 453

23

454

Supplementary Figure S6. Standard normalization curves for the quantitative transcriptomic 455

standard spike-ins. Dotplots denote log10 standards added (x-axis) vs. log10 standards 456

recovered (y-axis) for the five standards used. Different standards are denoted by colors: S3: 457

red, S5: blue, S6: green, S10: purple, S11: gold. 458

24

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