Characterizationof cyanobacterial glnAgenediversityandgeneexpression inmarine environmentsAngela H. Gibson1, Bethany D. Jenkins1, Frances P. Wilkerson2, Steven M. Short1 & Jonathan P. Zehr1

1Ocean Sciences, University of California Santa Cruz, Santa Cruz, CA, USA and 2Romberg Tiburon Center, San Francisco State University, Tiburon,

CA, USA

Correspondence: Angela Gibson, Earth and

Marine Sciences Building, University of

California, Santa Cruz, CA 95064, USA.

Tel: 11 831 459 4752; fax: 11 831 459 4882;

e-mail: agibson@ucsc.edu

Present address: Steven M. Short,

Department of Biological Sciences, University

of Denver, 2190 E. Iliff Avenue, Denver,

CO 80208, USA.

Received 23 May 2005; revised 18 August

2005; accepted 23 September 2005.

First published online 10 January 2006.

doi:10.1111/j.1574.6941.2005.00050.x

Editor: Riks Laanbroek

Keywords

glnA; glutamine synthetase; marine;

cyanobacteria; Synechococcus.

Abstract

PCR primers were designed and used to amplify glnA, the gene that encodes

glutamine synthetase, from pure cultures of cyanobacteria and four samples from

different marine environments. The glnA phylogeny was similar to that of the 16S

rRNA gene, indicating that glnA gene sequences can be used to identify

cyanobacteria expressing the glnA gene. Diverse unicellular cyanobacteria glnA

genes were recovered from the North Pacific Subtropical Gyre, Monterey Bay,

Chesapeake Bay and waters off the New Jersey coast. The majority of sequences

were closely related to sequences from Synechococcus strains (78–88% identical

DNA sequences). A few sequences that clustered with Prochlorococcus glnA genes

were recovered from Monterey Bay and the North Pacific Subtropical Gyre. The

expression of glnA was assayed by reverse transcriptase PCR to determine if there

was a daily pattern in gene expression of samples collected from New Jersey’s

Longterm Environmental Observatory site (LEO-15). glnA expression varied over

the day, with different glnA sequence types exhibiting different daily cycles. Results

showed that the glnA gene can be used to characterize the diversity of natural

populations of cyanobacteria, and to characterize gene expression patterns of

individual species or strains.

Introduction

Nitrogen is a major nutrient controlling primary productiv-

ity in marine environments (Sharp, 1983). The major forms

of inorganic nitrogen used by phytoplankton are nitrate and

ammonium. The use of different nitrogen compounds is the

basis for the model of regenerated and new production

(Dugdale & Goering, 1967), which is conceptually linked to

nitrogen and carbon export (Eppley & Peterson, 1979). It is

usually assumed that phytoplankton preferentially utilize

ammonium, because there are energetic costs of transport-

ing and using nitrate relative to ammonium. Ammonium

is assimilated by cyanobacteria primarily through the

glutamine synthetase–glutamine:2-oxoglutarate amido-

transferase (GS-GOGAT) pathway (Flores & Herrero, 1994;

Merrick & Edwards, 1995; Eisenberg et al., 2000; Herrero

et al., 2001). GS-GOGAT catalyses a two-step process in

which glutamine synthetase (GS) first converts glutamate to

glutamine in an ATP-dependent reaction and then gluta-

mate synthase (GOGAT) transfers the amide group of

glutamine to 2-oxoglutarate, producing two molecules of

glutamate in an NADPH-dependent reaction (Flores &

Herrero, 1994; Merrick & Edwards, 1995). Although another

pathway for directly incorporating ammonium into gluta-

mate exists, the glutamate dehydrogenase (GDH) pathway,

it has a high Km =�1 mM that is inefficient for cells growing

under ammonium limitation (Flores & Herrero, 1994). The

GS-GOGAT pathway has a higher affinity for ammonium

and is more efficient at NH41 assimilation when ammonium

concentrations are low (Flores & Herrero, 1994).

Although tracer experiments or biogeochemical measure-

ments can be used to determine community nitrogen

metabolism in different size classes of phytoplankton, the

use of molecular approaches (targeting DNA or mRNA) can

identify specifically which taxa or phylotypes are capable of

using different nitrogen sources (e.g. nitrogen, nitrate or, in

this case, ammonium), and which are actively expressing

genes for metabolism of individual compounds (Scanlan &

West, 2002). Genomic information shows that some cyano-

bacteria, such as Prochlorococcus strains MED4 and MIT

9313, do not have assimilatory nitrate reductase genes (Ro-

cap et al., 2002). And one marine Synechococcus isolate (MIT

S9220) from the Equatorial Pacific does not use nitrate as a

source of nitrogen (Moore et al., 2002). Thus, there are

FEMS Microbiol Ecol 55 (2006) 391–402 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

cyanobacterial communities that are composed of strains

that use only ammonium, or ammonium and nitrite, or all

three nitrogen compounds.

The objective of this study was to design glutamine

synthetase PCR primers to amplify cyanobacteria glnA

genes, in order to develop a molecular approach for

characterizing cyanobacterial populations and glnA gene

expression in marine systems.

Materials andmethods

Sample collection

Seawater samples for DNA analysis were collected from the

Choptank River and Chesapeake Bay samples from the R/V

Cape Henlopen in October 2000, and April and July 2001.

The lower Choptank River site (CT200) is located at

381460N, 761090W. The mid- and lower Chesapeake Bay

sites (CB200 and CB300) are located at 381340N, 761270W

and 371160N, 761090W, respectively. Samples were collected

from two Monterey Bay stations (M1 and M2) on 18

December 2001 [R/V Pt. Lobos, Monterey Bay Aquarium

Research Institute (MBARI) cruise no. 35201] and 22

February 2002 (R/V Pt. Lobos, MBARI cruise no. 2357).

The M1 and M2 mooring sampling locations are located at

36.751N, 122.031W, and 36.701N, 122.391W, respectively. In

order to see how the glnA gene is expressed by natural

populations, RNA samples were collected as part of a

collaborative Department of Energy (DOE)-supported pro-

gram (Bi-OMP) at the Longterm Environmental Observa-

tory site (LEO-15, located at 391270N, 741140W) on 22 July

2002, known as the Geochemical Rate-RNA Integration

Study, or ‘GRIST’ (Corredor et al., 2004). Samples for DNA

analysis were collected from the Hawai’i Ocean Time series

(HOT) site at station ALOHA (A Long Term Oligotrophic

Habitat Assessment, located at 221450N, 1581W), near

Hawai’i on 13 December 2002 (R/V Kilo Moana).

For the Monterey Bay samples, a Sea-Bird CTD (Sea-Bird

Electronics Inc., Kilo Moana Bellevue, WA) rosette of twelve

10 L Niskin bottles was used to collect samples from several

depths in the water column. Approximately 4–5 L of water

was prefiltered through 2.5 in diameter, 5 mm pore-size

Nytex filters held in a filter holder and attached to the

Niskin bottles with silicone tubing. Aliquots (500 mL) of the

prefiltered water were then filtered through 25 mm dia-

meter, 0.2mm pore-size Supor (Pall Gelman) filters. Each

filter was then stored in 250 mL of 1�TE buffer solution

(50 mM Tris-HCl, 50 mM EDTA) at � 80 1C. Samples were

collected from depths of 0–200 m (every 20 m) at the M1

and M2 stations. Additional Monterey Bay DNA extracts

from M1 (collected 22 February 2002) were provided by Dr

Ed DeLong (MIT).

Samples from the Chesapeake Bay, LEO and HOT (ALO-

HA) sites were collected by filtration of �1 L of seawater

through a SterivexTM capsule (Millipore, Bedford, MA)

using a peristaltic pump. Excess water was evacuated from

the filter by running air through the pump. Filter capsules

were stored at �80 1C for later analysis. Samples from

Chesapeake Bay were collected from the surface, mid-depth

and 1.5 m above the bottom at each station. Samples were

collected from a depth profile (0–200 m) at station ALOHA.

Only surface-water samples were collected at the LEO site.

Cyanobacteria cultures

In addition to the environmental samples, cultures of

cyanobacteria were obtained from the Pasteur Culture

Collection of Cyanobacteria, the Deutsche Sammlung von

Mickroorganismen und Zellkuturen GmbH, and the Agri-

culture Research Culture Collection. Synechococcus DNA

extracts (strains WH8008, WH8009, WH8104 and

WH8108) were also obtained for the study, courtesy of

Dr L. Campbell (Texas A1M). The Synechococcus strains

used in this study are given in Table 1. In order to cover a

broad range of cyanobacteria morphology and taxonomy,

other strains were used, including the filamentous hetero-

cystous (Tolypothrix sp. PCC7101, Nostoc sp. UTEX486 and

Anabaena sp. UTEX629) and the filamentous nonheterocys-

tous (Symploca sp. PCC8002, Lyngbya sp. UTEX7419).

DNAextraction

DNA was extracted from environmental and culture samples

using STE buffer [20% weight in volume (w/v) sucrose,

50 mM Tris-HCl, 50 mM EDTA] containing 5 mg mL�1

lysozyme, followed by proteinase K digestion (2 mg mL�1

final concentration), SDS addition (sodium dodecyl sul-

phate, 1% w/v final concentration) and PCI (phenol chloro-

form isoamyl alcohol 25 : 24 : 1) extraction as outlined in

(Steward et al., 2004). The ALOHA station samples were

extracted using the xanthogenate method described in

(Tillett & Neilan, 2000).

Primerdesign

To design glnA-specific primers, cyanobacterial and pro-

chlorophyte glnA DNA sequences, along with several of the

most closely related bacterial sequences from GenBank

(based on BLAST homology), were assembled and their

protein translations aligned (Fig. 1) using the Block Maker

algorithm (http://bioinformatics.weizmann.ac.il/Blocks/).

From the local alignment, regions of high conservation

among the cyanobacterial sequences were searched with the

CODEHOP (Consensus-Degenerate Hybrid Oligonuc-

leotide Primers) program, which generated several possible

primers at each block from a consensus of amino acid

FEMS Microbiol Ecol 55 (2006) 391–402c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

392 A.H. Gibson et al.

residues using an amino acid position-specific scoring

matrix (Rose et al., 1998). The best possible glnA primers

were designed from this consensus. The primers bracket a

region of the glnA gene that covers the latter half of the gene.

The outer pair are GLNcdhpCOF (50GACATCCGCACGAR

ATGYTNYT30) and GLNcdhpGOR (50CAATCGTAGTACA

TGGAGAATTCGTRNGGRTGN GG30), at positions 586

and 1409 bp, respectively, of the Synechococcus WH7803

glnA sequence. The inner primers are GLNcdhpCIF (50CG

GCGTCCCCATCGARAARCANCA30) and GLNcdhpGIR

Table 1. Characteristics of unicellular cyanobacteria cultured isolates (Boone et al., 2001; Rocap et al., 2002)

Genus Strain Cluster� PEw F, Mz Salinity O, C, B‰ Main characteristics

Prochlorococcus MED4 (CCMP1986) - Absent M - O High light adapted

Prochlorococcus SS120 (CCMP1375) - Absent M - O Low light adapted

Prochlorococcus MIT9313 - Absent M - O Low light adapted

Synechococcus WH8103 5.1 Present M High salt requirement O Motile, no chromatic adaptation

Synechococcus WH8102 5.1 Present M High salt requirement O Motile, no chromatic adaptation

Synechococcus WH8104z - - M - C -

Synechococcus WH8009z - - M - C -

Synechococcus WH8108z 5.1 Present M High salt requirement C Nonmotile, no chromatic adaptation

Synechococcus WH8008z - Present M - C -

Synechococcus WH7803 5.1 Present M High salt requirement B Nonmotile, no chromatic adaptation

Synechococcus WH5701 5.2 Absent M Halotolerant C Nonmotile

Synechococcus PCC6301 1.1 Absent F Low salt tolerance - -

Crocosphaera k WH8501 - Present M High salt requirement C -

Thermosynechococcus BP-1 - - F - - Thermophilic

Synechocystis PCC6803 2.1 Absent F Low salt tolerance - Motile, photoheterotrophic

�Cluster no. to which the strains are assigned in Bergey’s ManualwPE, phycoerythrin pigment.zF, freshwater species; M, marine species.‰O, oceanic; C, coastal; B, both.zStrains from which glnA was sequenced in this study.k Previously called marine Synechocystis

syncoWH7803 SEMLLMMAQLGIPIEKHHHELAGAGQsyncoWH8102 SEMLLLMGQLGIPTEKHHHEVAGAGQproMIT9313 SEMLLLMGQLGIPMEKHHHEVAGAGQproMED4 SEMLLQMAELGIPTEKHHHEVAGAGQSyncysPCC6803 TEMLLTMAAFGVPIEKHHHEVASGGQAnabaena TEMLLTMAKLGVPIEKHHHEVATGGQNostocPCC7120 TEMLLTMAKLGVPIEKHHHEVATGGQCaloPCC7601 TEMLLTMAACGVPIEKQHHEVATGGQSyncoPCC7942 SEMLLTMADCGVPIEKHHHEVATGGQGLN1_FRAAL SEMTRVLYETGITVEMQHHEVGTAGQFrankiaalni SEMTRVLYETGITVEMQHHEVGTAGQMycosmeg DQMATNLQNAGFTLERGHHEVGTAGQMycoleprae DKMLTNLTNAGFVLEKGHHEVGTGGQE.coli SEMSLVMEQMGLVVEAHHHEVATAGQ

outer inner

Cyanobacteria

Bacteria

syncoWH7803 DFINNWIDLKYE-EVQQLRQRPHPHEFTMYYDAsynco8102 DFIDNWIDLKYE-EVQQLRQRPHPHEFAMYYDAproMIT9313 DFIDNFIDMKYE-EVQQLRQRPHPHEFFMYYDAproMED4 DFIDNFIDMKYE-EVQQLRQRPHPHEFFMYYDASyncysPCC6803 DFVESWIEYKLDNEVNPMRLRPHPYEFSLYYDCAnabaena DFIQNWIDYKLANEVKQMQLRPHPYEFSIYYDVNostocPCC7120 DFIQNWIDYKLANEVKQMQLRPHPYEFSIYYDVCaloPCC7601 DFIQNWIEYKLVNEVKQLQLRPHPYEFYLYYDCSyncoPCC7942 DFIQNWIEYKLDNEVIPISLRPHPYEFALYYDCGLN1_FRAAL DLIETWLEYKRLNEVDAIRLRPHPYEFTLYYDIFrankiaalni DLIETWLEYKRLNEVDAIRLRPHPYEFTLYYDIMycosmeg DLIETWISYKRENEIMPIQIRPHPYEFSLYYDVMycoleprae DLIETWINFKRENEIEPVNIRPHPYEFALYYDVE.coli DEAIDAYIALRREEDDRVRMTPHPVEFELYYSV

Cyanobacteria

Bacteria

outer inner

196196196196196197197194196196196200200194

442442442442441442442439441441441446446438

REVERSE

FORWARD

Fig. 1. glnA gene cyanobacteria-specific nested

PCR primers designed with the CODEHOP

program (http://bioinformatics.weizmann.ac.il/

blocks/codehop.html) using cyanobacterial and

prochlorophyte glnA amino acid sequences

(dark arrow) and the most closely related bacter-

ial sequences (determined by BLAST homology).

Cyanobacterial sequences used were from Syne-

chococcus spp. (syncoWH7803, syncoWH8102,

syncoPCC7942), Prochlorococcus spp.

(proMIT9313, proMED4), Synechocystis sp.

(syncysPCC6803), Anabaena variabilis sp.

ATTCC29413 (Anabaena), Nostoc sp.

(NostocPCC7120) and Calothrix sp.

(CaloPCC7601). Bacterial species used in the

alignment were from Frankia alni CpI1 (Frankial-

ni), Mycobacterium smegmatis (Mycospmeg),

Mycobacterium leprae TN (Mycoleprae) and

Escherichia coli CFT073 (E. coli).

FEMS Microbiol Ecol 55 (2006) 391–402 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

393Characterization of cyanobacterial glnA gene diversity and gene expression

(50TCGTATTTGTAATCGATCCAGTTTTBNAYRAARTC30),

at positions 628 and 1334 bp, respectively, of the Synecho-

coccus WH7803 glnA sequence. The primers were synthe-

sized by Integrated DNA Technology Inc. (Coraville, IA).

The primers were tested against several noncyanobacter-

ial strains for specificity: Methanococcus sp. DSMZ 1224 and

Clostridium sp. NRRL B-527, Heliobacter sp. NRRL B-14647

and Sinorhizobium sp. NRRL L-45. The GlnA gene was not

amplified.

PCRamplification

The first-round PCR reactions (50mL) included 37.5mL

deionized water, 5mL of 10�ExTaqTM buffer solution

(Takara Bio Inc., Otsu, Shigu, Japan), 4 mL of 2.5 mM

dNTPs, 0.5 mL of 100mM GLNcdhpCOF primer, 0.5mL of

100mM GLNcdhpGOR primer and 0.5 mL ExTaqTM poly-

merase (5 UmL�1), along with 15–40 ng of genomic DNA

extract (2mL). All reaction mixes prior to addition of

ExTaqTM enzyme were filtered through a 100-kDa filter

(Millipore) at 5000 r.p.m. (3000 g). At least two negative

controls (no DNA extract) were included in every set of

reactions. The cycling times were one cycle at 94 1C for

5 min, followed by 30 amplification cycles (94 1C for 1 min,

50 1C for 1 min and 72 1C for 70 s) and a 7 min extension at

72 1C. A second round of PCR reactions (50mL) was repeated

using 2mL of the first reaction with 0.5mL of 100mM

GLNcdhpCIF primer and 0.5mL of 100mM GLNcdhpGIR

primer. Core mixes were filtered in the same manner as

described for the first round. The nested reactions were

amplified for one cycle at 94 1C for 5 min, followed by 30

cycles (94 1C for 1 min, 50 1C for 1 min and 72 1C for 1 min)

and ending with a 7 min extension at 72 1C.

RT-PCRamplificationofLEOsamples

RNA was collected at five time points taken at the LEO site

(07:04, 11:15, 16:00, 19:10 and 22:00 hours). RNA was

extracted using a RNeasys Mini Kit (Qiagen, Hilden,

Germany). A nested reverse transcriptase PCR (RT-PCR)

was performed using the glnA primers. For RT-PCR, a Super

ScriptTM One Step RT-PCR with the Platinums Taq DNA

Polymerase kit (Invitrogen Corp., Carlsbad, CA) was used

with 2mL (�13 ng) of RNA extract added to a 25mL reaction

for the first round. A control reaction was run without RT to

test for contaminating DNA. Two blank controls were

included with each reaction. The RT reaction mix consisted

of 9.5mL of sterile water filtered through a 5 kDa filter

(Millipore), 12.5 mL 2� buffer solution, 0.25 mL of 100 mM

GLNcdhpCOF primer, 0.25 mL of 100 mM GLNcdhpGOR

primer and 0.5 mL SuperscriptTM II RT. For the reaction mix

containing no RT the following were added: 9.9 mL of sterile

filtered water, 12.5mL 2� buffer, 0.25mL GLNcdhpCOF

primer, 0.25 mL GLNcdhpGOR primer and 0.1mL

Platinums Taq polymerase mix. The amplification protocol

was one cycle of 50 1C for 30 min and 94 1C for 2 min,

followed by 40 amplification cycles (94 1C for 30 s, 50 1C for

30 s, and 72 1C for 1 min), and a final 7 min extension step at

72 1C. The second round reaction was a 50 mL PCR reaction

containing 37.5mL water, 5 mL of 10� ExTaqTM buffer solu-

tion, 4mL of 2.5 mM dNTPs and 0.5 mL of 100mM

GLNcdhpCIF primer, 0.5 mL of 100 mM GLNcdhpGIR pri-

mer and 0.5 mL ExTaqTM polymerase (5 U mL�1), along with

2 mL of first round RT-PCR product. The amplification

protocol was one cycle at 94 1C, followed by 30 amplification

cycles (94 1C for 1 min, 50 1C for 1 min and 72 1C for 1 min)

and a 7 min extension cycle at 72 1C.

Cloningandplasmid isolation

PCR products from the second round were resolved on a 1%

agarose gel at �94 V. Gel fragments were excised and

purified using a QIAquick Gel Extraction Kit (Qiagen).

Amplification products were ligated and transformed using

a pGem T-system II vector kit (Promega Corp., Madison,

WI). Transformations were plated on LB-agar plates con-

taining 100 mg mL�1 carbenicillin disodium salt, 0.5 mM

isopropyl-b-D-thiogalactopyranosid (IPTG) and 80 mg mL�1

5-brom-4-chloro-3-indolyl-beta-D-galactopyranosid (X-

GAL) and incubated overnight at 37 1C. Individual colonies

were picked from the agar plates and grown overnight in

2–3 mL of LB broth and 100mg mL�1 ampicillin at 37 1C

with shaking (300–320 rpm). Either QIAprep Spin Miniprep

(Qiagen) or Montage Plasmid Miniprep (Millipore) kits was

used to purify the plasmids.

Sequencing

Purified plasmids were cycle sequenced using the ABI Prism

BigDye Terminator v3.0 Cycle Sequencing kit (Applied

Biosystems, Foster City, CA) and SP6 primer (Promega) for

27 cycles (96 1C for 10 s, 50 1C for 5 s and 60 1C for 4 min). A

Montage sequencing reaction cleanup kit (Millipore) was

used to clean up the sequence reactions. Products were

sequenced using an ABI Prism 310 or 3100 automated

sequencer (Applied Biosystems). The opposite strand was

sequenced using the T7 primer (Promega) for the represen-

tative sequences, which were submitted to GenBank under

accession numbers DQ020039–DQ020041, DQ020044–

DQ020056, DQ020058–DQ020061, DQ020064, DQ066752–

DQ066772, DQ070874–DQ070876, DQ070878–DQ070881

and DQ154120–DQ154134.

Phylogenetic analysis

DNA sequences were edited in GCG (Accelrys Inc., San

Diego, CA, USA) and translated. The Institute for Genomic

Research protein family sequences (TIGR-FAM) were

downloaded (www.tigr.org/TIGRFAMS) as a seed to create

FEMS Microbiol Ecol 55 (2006) 391–402c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

394 A.H. Gibson et al.

the HMMER alignment using the HMMR program in GCG.

The predicted amino acid sequences were then aligned with

HMMER in GCG, and then imported into the ARB software

package (Ludwig et al., 2004). The DNA sequences were

then aligned according to the protein alignment using ARB.

Neighbor-joining trees (Saitou & Nei, 1987) for both

protein and DNA alignments were generated with TREE-

CON for Windows (VanDePeer & Dewachter, 1994). The

phylogenetic distances between DNA sequences were calcu-

lated with a Jukes–Cantor distance correction in TREECON.

The Tajima–Nei correction was used for protein trees. Boot-

strap values were generated with 100 replicates for each tree.

The glnA-like sequence from the Archaea Thermoplasma

acidophilum (accession no. NC_002578) was used as the

outgroup to root both trees. Individual representative

sequences were selected from groups of sequences that

showed Z98% sequence similarity to construct the phylo-

genetic trees.

Results anddiscussion

glnA sequences fromcultivated isolates

Sequences of the glnA gene contained regions of conserved

amino acid sequences that allowed construction of cyano-

bacteria-specific CODEHOP primers for glnA (Fig. 1). The

specificity of the primers for cyanobacteria was checked by

testing the primers in reactions with DNA extracted from

cyanobacteria and heterotrophic bacteria (Fig. 2). None of

the heterotrophic bacterial strains amplified with the glnA

primers, indicating that the primers are selective if not

specific for cyanobacteria glnA. Of all subsequent amplifica-

tions of the glnA gene from environmental samples, only

two bacterial sequences were recovered.

The phylogenetic trees of glnA DNA and deduced amino

acid sequences had several features that are consistent with

16S rRNA and, rpoC1 gene phylogenies (Ferris & Palenik,

1998; Moore et al., 1998; Toledo et al., 1999; Rocap et al.,

2002; El Alaoui et al., 2003; Rocap et al., 2003). The

Synechococcus and Prochlorococcus glnA gene sequences form

deeply divergent clusters and are well separated from each

other (Fig. 3). The glnA sequences from cultures of Nostoc

UTEX486, Anabaena sp. UTEX629 and Tolypothrix sp.

PCC7101 clustered with sequences from other heterocystous

cyanobacteria (Fig. 3). The glnA sequence from Nostoc sp.

UTEX486 was 89% identical (on the DNA level) to the glnA

gene from the heterocystous filamentous cyanobacterium

Nostoc punctiforme.

The glnA sequences from other unicellular (Synechococcus

elongatus sp. PCC7942, NC004113; Synechocystis sp. PCC6803,

NC000911) and filamentous nonheterocystous (Symploca sp.

PCC8002, DQ020034; Lyngbya sp. PCC7419, DQ020037;

Trichodesmium erythraeum, NZ_AABK0300000000) cyano-

bacteria form deep branches (Fig. 3), consistent with 16S

rRNA gene phylogeny (Wilmotte, 1994).

Sequences from Synechococcus spp. WH8009, WH8104

and WH8108 (95–97% identical to each other) form a

group that is closely related to the cluster containing

sequences from the motile Synechococcus spp. WH8102 and

WH8103 (accession numbers NC005070 and AF027611,

respectively). Sequences from Synechococcus spp. WH8008

and WH7803 (DQ020031 and AF026393, respectively) form

a separate group in both DNA and amino acid trees (Fig. 3,

amino acid tree not shown). The Synechococcus sp. WH8102

is considered to be representative of oceanic strains (Water-

bury et al., 1986). Synechococcus spp. WH8108 and WH8008

were isolated from coastal regions and Synechococcus sp.

WH7803 is found both in oceanic and in coastal environ-

ments (Table 1) (Toledo & Palenik, 1997; Herdman et al.,

2001; Rocap et al., 2002; Scanlan & West, 2002). The coastal

and oceanic strains WH8102, WH8103 and WH7803 form a

genetically coherent group classified as marine cluster A (in

700bp

MW

Mar

ker

Posi

tive

Con

trol

Bla

nk

Neg

ativ

e C

ontr

ol

CB

300

(0m

)

CB

300

(3.8

m)

MW

Mar

ker

Bla

nk

Fig. 2. Ethidium-bromide-stained agarose gel of glnA PCR amplification

products amplified from environmental samples (Chesapeake Bay, sta-

tion CB300, at 0 and 3.8 m water depth). DNA from a cultured

cyanobacteria Tolypothrix (PCC7101) and a heterotrophic bacteria Sino-

rhizobium (NRRL L-45) were used as positive and negative controls,

respectively. The amplified glnA fragments are approximately 700 bp in

size, as determined from the molecular weight markers (MW Marker).

FEMS Microbiol Ecol 55 (2006) 391–402 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

395Characterization of cyanobacterial glnA gene diversity and gene expression

0.1 substitutions site

Thermoplasma acidophilum NC_002578

LE3473P25* LEO 0m DQ070878

HT9013P42* (4) HOT 45m DQ070875MB3482P1* (7) M1 0m DQ066772

Lyngbya PCC7419 * DQ020037

Anabaena UTEX629 * DQ020038CT921P75* (4) CT200 0.9m DQ066762

Prochlorococcus CCMP1375 NC005042

MB2318P91* M1 100m DQ070874

Nostoc PCC7120 NC003272

Synechococcus WH8008 * DQ020031

Symploca PCC8002 * DQ020034

HT9013P7* HOT 45m DQ066766

HT9013P44* HOT 45m DQ066769

Thermosynechococcus elongatus BP-1 NC004113

Synechococcus WH8108 * DQ070877

CT914P20* CT200 7.9m DQ066757

HT9013P11* HOT 45m DQ066767HT9013P10* HOT 45m DQ066768

Synechococcus WH8104 * DQ020033Synechococcus WH8009 * DQ020032

HT9015P18* (2) HOT 25m DQ066770HT9013P53* HOT 45m DQ070876

Synechococcus WH8103 AF027611Synechococcus WH8102 NC005070

LE3473P30* (22) LEO 0m DQ070879MB2308P17* (141) M2 200m DQ066771

CB912P13* (2) CB300 15m DQ066753LE3472P5* (33) LEO 0m DQ070880

CT923P64* (15) CT200 7.9m DQ066760CB912P2* (53) CB300 15m DQ066754

CB912P10* (4) CB300 15m DQ066755 LE3472P2* (3) LEO 0m DQ070881

CT914P26* (4) CT200 7.9m DQ066758CB900P33* (3) CB300 15m DQ020061

Synechococcus WH5701 AF027612

CT912P15* CB300 15m DQ066752 Synechococcus WH7803 AF026393

CT923P68* CT200 7.9m DQ066759CT914P10* (2) CT200 7.9m DQ066756

HT9007P10* (2) HOT 125m DQ066765Prochlorococcus MIT9313 NC005071

Prochlorococcus MED4 BX572091Prochlorococcus CCMP1986 NC005072

CT923P66* CT200 7.9m DQ066761Synechococcus elongatus PCC6301 NC006576

CB900P38* (4) CB300 15m DQ020064 Synechocystis PCC6803 NC000911

CT921P84* (2) CT200 0.9m DQ066763CT921P73* CT200 0.9m DQ066764

Crocosphaera watsonii WH8501 AADV01000000Trichodesmium erythraeum IMS101 Z AABK0300000000

Tolypothrix PCC7101* DQ020035 Calothrix sp. L05609

Nostoc UTEX486 * DQ020036 Nostoc punctiforme ATCC29133 AAAY02000000

Anabaena variabilis ATCC29413 NZ AAEA01000001 Anabaena azollae AJ49658

100

98

90

73

97

61

70

60

100

97

51

99

59

100

88

57

100

100

100

100

100

100

50

100

51

93

100

100

100

100

53

100

87

100

99

98

100

88

100

100

77

100

80

100

Fig. 3. Neighbor-joining phylogenetic tree (DNA) showing the different glnA clusters. Environmental sequences are indicated with their station and

water depth on the right. Cultures and environmental samples that were sequenced in this study are indicated by an asterisk (�). The tree was generated

with TREECON as described in the Methods section. Bootstrap values were generated from 100 replicates for each tree. The sequence from

Thermoplasma acidophilum (accession no. NC_002578) was used to root the tree. Genbank accession numbers are indicated. The number of

sequences for each representative sequence is shown in parentheses.

FEMS Microbiol Ecol 55 (2006) 391–402c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

396 A.H. Gibson et al.

Boone et al., 1989, p. 721; cluster 5.1 in Boone et al., 2001, p.

721) and are characterized by having C-phycoerythrin as

their major light-harvesting pigment (Herdman et al., 2001;

Rocap et al., 2002; Scanlan & West, 2002). In addition,

Synechococcus spp. WH8102 and WH8103 form a distinct

subgroup within group A and are not capable of chromatic

adaptation (Scanlan & West, 2002).

Prochlorococcus glnA gene sequences form separate

branches from the Synechococcus glnA gene sequences, with

deep divergence between sequences of the Prochlorococcus

sp. MIT9313 and Prochlorococcus sp. MED4 low- and high-

light ecotypes, respectively. The sequences from Prochloro-

coccus spp. MED4 and CCMP1986 are identical as they are

the same strain maintained in different culture collections.

Sequences from Prochlorococcus spp. MIT9313, MED4

(also CCMP1986) and CCMP1375 (also SS120) form two

divergent groups on the basis of rpoC1 or rRNA intergenic

spacer regions (Toledo & Palenik, 1997; Ferris &

Palenik, 1998; Rocap et al., 2003), which is consistent with

the light adaptation phenotypes of MIT9313 and

CCMP1375 (SS120) (low-light) and MED4 (high-light).

CCMP1375 (SS120) and MIT9313 consistently cluster

together on the basis of molecular markers, including the

glnA gene.

Overall, the clustering of representative cyanobacterial

glnA sequences was similar between the DNA and amino

acid trees (data not shown), but there is greater sequence

divergence in the former, as would be expected. The

similarity of topologies of the phylogenetic trees of the glnA

gene and other genes provides a way to classify uncultivated

cyanobacteria that express the glnA gene.

Cyanobacterial glnAdiversity inmarineplankton

glnA sequences were amplified from a variety of marine

environments, including a coastal shelf (coastal environ-

ments on the west and east coast of North America, open

ocean and an estuary), to test the ability of the primers to

amplify the glnA gene from different marine environments.

Different phylogenetic groups of glnA sequences were re-

covered from the different sites.

The Chesapeake Bay estuary (including the Choptank

River) had a diverse set of glnA sequences that were closely

related to sequences from the filamentous heterocystous

cyanobacteria genera, Anabaena and Nostoc, and other

sequences that clustered among the divergent cyanobacterial

glnA sequences from Synechococcus elongatus sp. PCC6301

and Synechocystis sp. PCC6803 (Table 2, Fig. 3). It was the

only site studied that had the glnA gene from these groups of

cyanobacteria, consistent with the influence of freshwater

inputs. The Chesapeake Bay estuary is fed by a number of

rivers, including the Choptank River, that influence salinity,

nutrient concentrations and turbidity (Fisher et al., 1988;

Heidelberg et al., 2002). Filamentous cyanobacteria, in

particular heterocystous species, are commonly found in

freshwater and brackish environments (Herdman et al.,

2001).

All of the glnA sequences recovered from the

North Pacific Subtropical Gyre station ALOHA of the

HOT program, from Monterey Bay and from the LEO

site clustered with Synechococcus and Prochlorococcus

sequences.

Some of the glnA sequences from the Choptank River and

the Chesapeake River grouped with Synechococcus as well,

but formed separate clusters from the sequence groups from

oceanic samples (Monterey Bay, Station ALOHA) (Fig. 3).

The Choptank River and Chesapeake Bay glnA sequences

were most closely related to Synechococcus sp. WH8008,

WH7803 and WH5701 (Table 2, Fig. 3). The glnA sequences

from the LEO site clustered alone (LE3473P25) or with

Chesapeake Bay (LE3472P2, LE3472P5) or Monterey Bay

(LE3473P30) sequences. Most of the Monterey Bay Synecho-

coccus glnA sequences clustered with this LEO LE3473P30

sequence type. Some of the Monterey Bay sequences were

within the Synechococcus cluster but distant from either of

the large clusters containing the oceanic or estuarine glnA

types (MB3482P1).

Synechococcus glnA sequences from the oceanic site (Sta-

tion ALOHA of the HOT program) clustered with Synecho-

coccus sequences (Synechococcus sp. WH8102, WH8103,

WH8009, WH8104) (Table 2, Fig. 3), but formed several

small clusters that may represent several ecotypes or strains

(Fig. 3). Isolates WH8102 and WH8103 are in the marine

cluster of Synechococcus known as group A (or MC-A),

which is characterized by an elevated salt requirement,

C-phycoerythrin and nonflagellated motility (Toledo et al.,

1999; Moore et al., 2002; Scanlan & West, 2002) (Table 1).

These strains are abundant in the oligotrophic regions of the

open ocean and are generally not found in coastal regions

(Waterbury et al., 1986). Thus, the recovery of glnA

sequences similar to those of Synechococcus sp. WH8102 is

consistent with the expected phylotypes at the oligotrophic

ocean station ALOHA.

Prochlorococcus glnA sequences were only detected in

Monterey Bay and at station ALOHA. The Prochlorococcus-

like sequences recovered from station ALOHA branched

most closely with the sequence from Prochlorococcus sp.

MIT9313 (Table 2, Fig. 3), the low-light-adapted phylotype

(Moore et al., 1998, 2002; El Alaoui et al., 2003). The

sequences (DNA 97% identical to Prochlorococcus sp.

MIT9313) were obtained from a water depth of 125 m,

below the chlorophyll maximum at about 85–90 m (0.21

mg L�1) and within the nitracline at about 80 m. Only a few

samples and sequences from station ALOHA were analysed

for the glnA gene in this study and no Prochlorococcus

sequences were recovered from depths shallower than

FEMS Microbiol Ecol 55 (2006) 391–402 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

397Characterization of cyanobacterial glnA gene diversity and gene expression

125 m. Therefore, it was not possible to conclude whether

there was a depth distribution of low- and high-light-

adapted phylotypes based on the glnA sequences analysed

here.

Prochlorococcus abundance always exceeds Synechococcus

abundance by two orders of magnitude at station ALOHA

(Campbell et al., 1997). Only 15 glnA sequences were

recovered, two of which were Prochlorococcus. The low

recovery of Prochlorococcus glnA relative to Synechococcus at

the open ocean site (station ALOHA) suggests primer bias,

or differences in extraction efficiency, but only a relatively

low number of sequences were analysed in this study.

Two Prochlorococcus sp. glnA sequences were recovered

from Monterey Bay. The two Prochlorococcus-like glnA

sequences (one partial sequence not shown in the glnA tree)

were recovered from 100 m at M1 in December 2001,

clustering with glnA from Prochlorococcus sp. MIT9313.

The recovery of this sequence from a deep sample is

consistent with the fact that Prochlorococcus sp. MIT9313 is

adapted to low-light conditions (Moore et al., 2002; Scanlan

& West, 2002; El Alaoui et al., 2003). The recovery of only a

few Prochlorococcus glnA sequences is consistent with the

low abundance of Prochlorococcus in Monterey Bay and

coastal environments in general.

Table 2. Representative sequences recovered from the different environmental sites, showing most closely related sequences in Genbank on the basis

of DNA or deduced amino acid sequences

Sample Representative sequence�

No. of sequences Z98%w

Closest isolate based on BLAST

% Similarity

DNA Protein DNA Protein

Chesapeake CB900p33 3 3 Synechococcus WH5701 82 87

Chesapeake CB900p38 4 4 Synechocystis PCC6803 85 98

Chesapeake CB912p2 53 53 Synechococcus WH8008 98 99

Chesapeake CB912p10 2 - Synechococcus WH8009 83 -

Chesapeake CB912p13 2 2 Synechococcus WH8009 85 92

Chesapeake CB912p14 2 4 Synechococcus WH8104 84 88

Chesapeake CT914p10 2 2 Synechococcus WH8009 85 93

Chesapeake CT914p26 4 4 Synechococcus WH5701 81 86

Chesapeake CT914p27 2 - Synechococcus WH8009 84 -

Chesapeake CT921p75 4 4 Anabaena azollae 86 93

Chesapeake CT921p84 2 2 Symploca PCC8802 75 85

Chesapeake CT923p64 13 15 Synechococcus WH8009 82 94

HOT HT9007p10 2 2 Prochlorococcus MIT9313 97 98

HOT HT9013p42 4 4 Synechococcus WH8009 82 91

HOT HT9015p18 2 2 Synechococcus WH8104 88 97

LEO LE3472p2 3 3 Synechococcus WH8104 83 90

LEO LE3472p5 31 31 Synechococcus WH8009 85 93

LEO LE3472p11 5 6 Synechococcus WH8102 82 95

LEO LE3473p29 6 8 Synechococcus WH8102 82 95

LEO LE3473p30 3 3 Synechococcus WH8102 82 95

LEO LE3473p34 2 2 Synechococcus WH8009 84 90

LEO LE3474p48 5 - Synechococcus WH8102 81 -

LEO LE3474p85 4 6 Synechococcus WH8102 81 96

Monterey Bay (2001) MB2308p17 65 71 Synechococcus WH8102 81 94

Monterey Bay (2001) MB2311p5 2 - Synechococcus WH8102 81 -

Monterey Bay (2001) MB2318p8 2 - Synechococcus WH8102 82 -

Monterey Bay (2001) MB2318p15 2 2 Synechococcus WH8102 81 91

Monterey Bay (2001) MB2324p1 2 - Synechococcus WH8102 83 -

Monterey Bay (2001) MB2324p3 2 2 Synechococcus WH8102 81 94

Monterey Bay (2002) MB3482p1 7 7 Synechococcus WH8009 81 92

Monterey Bay (2002) MB3483p25 10 - Synechococcus WH8102 80 -

Monterey Bay (2002) MB3484p33 12 12 Synechococcus WH8102 82 93

Monterey Bay (2002) MB3484p47 7 - Synechococcus WH8102 83 -

Monterey Bay (2002) MB3486p73 9 9 Synechococcus WH8102 81 95

Monterey Bay (2002) MB3486p76 2 2 Synechococcus WH8102 82 94

Monterey Bay (2002) MB3488p90 26 45 Synechococcus WH8102 82 94

�Two or more sequences withZ98% in nucleotide sequence similarity.wTotal number of sequences withZ98% similarity to the representative sequence.

FEMS Microbiol Ecol 55 (2006) 391–402c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

398 A.H. Gibson et al.

ExpressionofglnA

The use of glnA to characterize cyanobacteria populations

expressing the glnA gene was tested in a collaborative study

at the Rutgers University LEO site from 20 to 22 July 2002.

The objective of this collaborative experiment was to

investigate the relationship between gene expression and

biogeochemical processes (the ‘GRIST’ study, Corredor

et al., 2004). glnA gene expression was detected over the diel

experiment by RT-PCR (Fig. 4). The greatest amplification

occurred at 11 : 15, followed by the 16 : 00 and 22 : 00 time

points (Fig. 4). The RNA samples did not amplify when RT

was not added, demonstrating that the RT-PCR results were

derived from mRNA and not contaminating genomic DNA,

and there was no amplification in the negative controls (Fig.

4). This is consistent with a diel study of rbcL transcripts in

the GRIST experiment (Corredor et al., 2004), in which rbcL

mRNA peaked during the morning hours (Corredor et al.,

2004). However, the RT-PCR in this case was not quantita-

tive, and the pattern of amplification over the diel cycle

should be interpreted with caution.

Five different glnA sequence groups were identified from

the amplification products (Figs 5 and 6), but all clustered

with sequences from Synechococcus spp. (strains WH8102,

WH8104 or WH8009; Table 2). Most of the 68 sequences

were in group 4. Sequences in this group were recovered

from all time points, but most of them were recovered in the

morning (Fig. 5). The majority of the sequences obtained by

RT-PCR from samples collected at the LEO site formed a

cluster with sequences that had also been obtained from the

Chesapeake Bay and Choptank River, and distantly grouped

with sequences from Synechococcus spp. WH8009, WH8102

and WH8104 (81–85% identical). Some of the LEO se-

quences also grouped closely with the larger cluster of

Monterey Bay clones, although all were less than 98%

identical to the Monterey Bay sequences (Table 2, Fig. 3).

This indicates that although the LEO samples group with

sequences from other coastal environments, they are distinct

from the Monterey Bay Synechococcus-like sequences.

There were shifts in relative abundances of glnA sequence

types over the diel cycle (Fig. 5). Although the number of

700bp

700bp

Bla

nk

22:0

0

19:1

0

16:0

0

11:1

5

07:0

4

Bla

nk

MW

Mar

ker

(a)

(b)

Fig. 4. Ethidium-bromide-stained agarose gel of the second-round

nested reverse transcriptase (RT-)PCR amplification of glnA (a), and

controls without RT added to test for contaminating DNA (b). The gel

shows RT-PCR amplification products from five time points over a daily

cycle at the LEO-15 site. glnA fragments are �700 bp in size.

Time

0:00 8:00 16:00 22:00

Sal

init

y

31.75

31.80

31.85

31.90

31.95

32.00

32.05

Tem

pera

ture

(°C

)

15.5

16.0

16.5

17.0

17.5

18.0

18.5Group

1

5

4

3

2

7:04 11:15

16:00 19:00 22:00

2

4

6

2

7

5

19

6

1122

514

Fig. 5. Temperature (m) and salinity (�) over a

daily cycle starting on 22 July 2002 at LEO-15

in relation to the composition of glnA clone

libraries derived from reverse transcriptase PCR

of RNA collected at different times during the

day. Pie charts show the number of sequences

of each phylogenetic type (groups 1–5, indi-

cated on the phylogenetic tree in Fig. 6)

recovered over the cycle.

FEMS Microbiol Ecol 55 (2006) 391–402 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

399Characterization of cyanobacterial glnA gene diversity and gene expression

0.1 substitutions site

LE3476P84 22:00 DQ154129LE3472P75 7:04 DQ154130LE3472P5 (26) (13) 7:04, (4) 11:15, (1) 16:00, (5) 19:00, (3) 22:00 DQ070880

LE3476P91 22:00 DQ020055

LE3473P25 11:15 DQ070878

LE3476P81 22:00 DQ020052

LE3473P34 11:15 DQ154132

LE3476P93_1 22:00 DQ020059

LE3475P89 19:00 DQ020050

LE3474P55 16:00 DQ020048

LE3474P41 16:00 DQ154128

LE3473P28 11:15 DQ154133

LE3474P42 16:00 DQ020041

LE3474P83 16:00 DQ154120

LE3476P92 22:00 DQ020056LE3476P83 22:00 DQ020053

LE3474P48 16:00 DQ154121 LE3474P47 16:00 DQ020046

LE3473P76 11:15 DQ020040LE3473P32 (4) (3) 11:15, (1) 16:00 DQ154122

LE3476P93 22:00 DQ020058LE3474P49 16:00 DQ154123

LE3473P37 11:15 DQ154124LE3473P30 11:15 DQ070879

LE3476P90 22:00 DQ020054LE3476P85 22:00 DQ154125LE3472P14 7:04 DQ154126LE3472P11 7:04 DQ154127

LE3476P95 22:00 DQ020060LE3474P44 (3) 16:00 DQ020044

LE3474P53 16:00 DQ020047LE3473P39 11:15 DQ020039

LE3475P90 19:00 DQ020051LE3475P87 19:00 DQ154131

LE3475P86 19:00 DQ020049LE3474P46 16:00 DQ020045

LE3472P4 7:04 DQ154134LE3472P2 7:04 DQ070881100

98

68

100

50

80

100

69

98

82

100

93

73

60

65

99

91

75

88

100

60

81

66

100

74

100

100

98

100

99

100

51

75

59

85

97

93

100

98

100

100

90

88

100

100

62

100

66

100

Prochlorococcus CCMP1375 NC005042Prochlorococcus MED4 BX572091Prochlorococcus CCMP1986 NC005072

Prochlorococcus MIT9313 NC005071

Synechococcus WH8008

Synechococcus WH8103 AF027611Synechococcus WH8102 NC005070

Synechococcus WH8104Synechococcus WH8009

Synechococcus WH8108

Synechococcus WH5701 AF027612

Synechococcus elongatus PCC6301 NC006576Thermosynechococcus elongatus BP-1 NC004113Synechocystis PCC6803 NC000911

Trichodesmium erythraeum IMS101 NZ AABK0300000000

Anabaena UTEX629Anabaena azollae AJ49658Nostoc PCC7120 NC003272Nostoc punctiformes ATCC29133 AAAY02000000

Nostoc UTEX486 DQ020036 Calothrix sp. L05609Tolypothrix PCC7101 DQ020035

Lyngbya PCC7419 DQ020037Symploca PCC8002 DQ020034

Thermoplasma acidophilum NC_002578

1

4

5

2

3

Fig. 6. Neighbor-joining glnA phylogenetic tree (based on DNA sequences) showing the different glnA clusters for the LEO-15 site samples recovered

by reverse transcriptase PCR. The tree was generated with the TREECON software program as described in the Methods section. Bootstrap values were

generated from 100 replicates for each tree. The sequence from Thermoplasma acidophilum (accession no. NC_002578) was used to root the tree. The

number of sequences obtained for each representative sequence is shown in parentheses. The major glnA groups for the LEO samples are numbered

1 to 5 (see Fig. 5).

FEMS Microbiol Ecol 55 (2006) 391–402c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

400 A.H. Gibson et al.

clones that were sequenced from each time point was

relatively small, the data indicate that different species or

strains of Synechococcus are expressing glnA at different

times over the daily cycle.

Summary

Primers for amplification of cyanobacterial glnA were devel-

oped that amplified glnA from a variety of cyanobacteria

(including Prochlorococcus, filamentous and freshwater spe-

cies). These primers also successfully amplified glnA from a

number of aquatic environments. The majority of sequences

amplified from the aquatic environments using the glnA

primers were closely related to sequences from Synechococ-

cus culture isolates, consistent with the known abundance

and importance of this group in aquatic environments. A

few sequences that were closely related to Prochlorococcus

glnA were detected in oceanic environments, demonstrating

that these primers could be useful in characterizing both

Prochlorococcus and Synechococcus populations in the envir-

onment. However, the reasons for the low recovery of

Prochlorococcus glnA genes at station ALOHA are unknown,

but suggest either primer bias or differential extraction

efficiency.

The primers were shown to be useful in detecting patterns

of glnA expression by RT-PCR. The diversity of glnA

transcripts differed with time of day, indicating that differ-

ent species have different daily patterns of glnA gene

expression. Gene expression studies will have to take time

of day into account in order to assess the diversity of

populations actively expressing glnA. The results show that

glnA can provide a phylogenetic and functional gene marker

for basic nitrogen metabolism in cyanobacteria, and when

combined with markers for other nitrogen-utilization path-

ways, can provide a means for determining the nitrogen

metabolic pathways in natural populations of marine cya-

nobacteria.

Acknowledgments

We are grateful to all of the students and postdoctoral

researchers in the laboratory for help in research and

manuscript preparation. Special thanks go to Dr Bess Ward

for providing the Chesapeake Bay samples, Dr Lisa Camp-

bell for providing DNA from cultivated cyanobacteria and

Dr Deborah Bronk for providing nitrogen chemistry data.

This project was supported by the DOE Bi-OMP program.

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