Characterization of nitrogenase gene distribution and activity in WCA-2A periphyton Puja Jasrotia Image source: .

Characterization of nitrogenase gene

distribution and activity in WCA-2A periphyton

Puja Jasrotia

Image source: http://www-cyanosite.bio.purdue.edu

Why is Nitrogen important

• A major constituent of living cells.

• Nitrogen gas (N2) makes up 79% of the atmosphere.

• Required by all living organisms, but only a few can fix it.

• Often the limiting nutrient in wetlands.

Biological Nitrogen fixation

• N2 NH4+

• Catalyzed by nitrogenase enzyme

• Only prokaryotic organisms can fix nitrogen.

• Induced as a response to low conc. of fixed N2. High conc. of O2 and NH4

+ repress nitrogenase synthesis

VEGETATION :

U3

Lake

Okeechobee

WCA -1

WCA - 3

Everg

lades

Agricu

ltura

l Are

a

WCA -2A

0 1 2 3 4 5

Km

Everglades

National Park

Cattail

Sawgrass/slough

Cattail/ sawgrass mix

F1F4

Nutrient Transect in WCA-2A

Cattail ~ 1500 mg P/Kg

Sawgrass

~ 500 mg P/Kg

Florida Everglades

Periphyton processes

• Metabolically diverse microbial composition contributes to mat’s internal nutrient cycling.

• Important source of fixed N and C.

• Structural and functional changes indicative of nutrient enrichment.

• Contributes approx 10g N m-2y in oligotrophic WCA2A (Inglett et al., 2004).

• Lack of molecular characterization of diazotrophs.

• Identify diazotrophic groups for a broad understanding of

• nutrient limitation • factors regulating growth • indicator species of eutrophication and • flow of N in periphyton

Objectives

1. Compositional analysis of diazotrophs along the nutrient gradient in WCA-2A based on nifH diversity.

2. nifH gene expression during a diel cycle.

3. Spatial distribution of pmoA defined diversity in floating periphyton along the nutrient gradient.

Hypotheses

1. Periphyton nifH composition differs between eutrophic and oligotrophic regions as a result of relative N limitation.

2. A shift in the most active nitrogen fixing groups will be observed throughout a diel cycle.

3. pmoA diversity will vary with nutrient concentrations.

Work planPeriphyton sample Epiphyton sample

Methane oxidation

N2 fixation

Community assemblage composition analysis

pmoA phylogenetic analysis by sequence analysis

nifH phylogenetic analysis by sequence analysis

N2 fixation

Functional gene

Analysis

RT-PCR - characterizing nifH transcripts

1. Spatial variability of nifH diversity in periphyton

Eutrophic site

Oligotrophic site

Floating periphyton mats

Phylogenetic tree

Periphyton Nucleic acid

PCR amplification

Cloning

SequencingSeq. Analysis

RFLP

Image source: www.rothamsted.bbsrc.ac.uk

Rarefaction analysis

0

2

4

6

8

10

12

14

16

18

0 20 40 60 80 100 120 140 160

No. of clones analyzed

No. of OTU

F1

F4

U3

Rarefaction analysis of nifH clones from F1, F4 & U3.

0.1 substitutions/site

Frankia sp. CcI3

Uncultured bacterium NR1636

F1-10 F1-7 F1-2

Anabaena sp. I1

Fischerella UTEX1903

U3-8

Halothece sp. MPI 96P605

F4-9

Nodularia sp. KAC 13

Uncultured bacterium NR1629

U3-4

F1-8

U3-29

U3-12

F1-4

F1-1

Pseudanabaena PCC7403

U3-25

U3-14

Fischerella UTEX1931

Nostoc PCC 6720 Anabaena variabilis

Anabaena 7120 Anabaena sp. A2

Anabaena sp. L-31 U3-17

Scytonema sp.Nostoc muscorum

Nostoc sp. Nostoc commune (UTEX 584)

F1-5 F1-3

F1-9 F1-6

F4-6 F4-5

F4-4 Calothrix sp. Dermocarpa sp.

Chroococcidiopsis thermalis PCC 7203

Symploca sp. PCC 8002 Microcoleus chthonoplastes

Myxosarcina sp. Xenococcus sp.

Gloeothece sp. Synechocystis sp.

Cyanothece sp. WH 8902 Cyanothece ATCC51142

U3-16 U3-10

Plectonema boryanum (IU 594) F4-8

Uncultured bacterium clone BSC-2 F4-7

F4-10 F4-3

U3-18 U3-1

U3-13 U3-15

F4-12 F4-11

U3-6 U3-5

U3-9 U3-11

F4-2 F4-1 U3-28

U3-7 Marine stromatolite eubacterium

Lyngbya lagerheimii (UTEX 1930)U3-26 U3-3

U3-27 U3-23

U3-24 U3-19

U3-21 U3-2

U3-22 U3-20

Phormidium sp. Phormidium sp. AD1

100

81

100

82

64

100

95

100

51

60

53

93

71

64

58

94

100

68

77

98

88

100

61

90

100

100

100

93

75

100

89

87

63

62

100

100

100

90

100

100

56

100

67

100

100

60

95

80

83

100

100

89

100

61

Phylogenetic tree of cyanobacterial nifH clones from F1, F4 and U3.

Heterocystous cluster

Non-heterocystous unicellular cluster

Non-heterocystous cluster

Unidentified cluster

Non-heterocystous cluster

Unidentified cluster

0.1 substitutions/site

Frankia sp. CcI3

Spirochaeta zuelzerae

Uncultured bacterium clone 36_Z65C

F4-1

Uncultured nitrogen-fixing bacterium B3

Azotobacter chroococcum MCD 1

Methylomonas methanica clone 68-1

U3-5

Uncultured bacterium clone MO175A10

U3-3

Desulfovibrio vulgaris

Desulfovibrio africanus

F1-4

Methylosinus trichosporium

F4-3

Methylocystis echinoides clone 2-8

F1-3

F1-2

Uncultured bacterium clone Yushu-12

Uncultured bacterium clone SJY-31

U3-12

U3-10

Spirochaeta aurantia clone 2

F4-4

Uncultured bacterium clone CB907H81

U3-7

Desulfosporosinus orientis

Uncultured eubacterium 'Ice aggregate'

U3-8

U3-6

Klebsiella variicola strain T29A

Klebsiella pneumoniae

U3-11

U3-9

U3-13

U3-2

U3-4

U3-1

Rhodopseudomonas sp. HMD89

Methylobacterium sp. xct7

Methylocystis methanolicus clone 10-5

Methylocystis sp. LW5

Methylocella silvestris

F1-1

Rhizobium sp.

Bradyrhizobium sp. IRBG 230

Uncultured bacterium clone 31A-2n

F4-2

100

56

100

94

73

98

56

80

70

100

58

57

100

100

76

80

77

100

100

68

85

100

100

58

57

100

55

99

Phylogenetic tree of proteobacterial nifH clones from F1, F4 & U3.

-proteobacteria

-proteobacteria

-proteobacteria

Distribution of nifH clones from F1, F4 & U3

F1

Unicellular cyanobacteria (Subsection II)

7%

Heterocystous cyanobacteria (Subsection III)

54%

-proteobacteria35%

proteobacteria4%

F4

Non heterocystous cyanobacteria (Subsection III)

2%

Heterocystous cyanobacteria (Subsection IV)

5%

-proteobacteria9%

-proteobacteria5%

Novel cyanobacterial

cluster79%

U3

-proteobacteria2%

Novel cyanobacterial

cluster (2)12%

Novel cyanobacterial

cluster (1)18%

Non heterocystous cyanobacteria (Subsection III)

9%

Heterocystous cyanobacteria (Subsection IV)

16%

Novel proteobacterial

cluster5%

-proteobacteria12%

-proteobacteria26%

Spatial and temporal interactions

Image source: www.botany.hawaii.edu

Spatial and temporal interactions between

oxygenic photosynthesis and N2 fixation Night timeDaytime

Mat Surface

Anaerobic N2 Fixation

Oxygenic Photosynthesis

Aerobic N2 Fixation

Anoxygenic Photosynthesis

Anaerobic N2 Fixation

Organic Carbon

Pool

OxicAnoxic

Oxic

AnoxicOrganic C

Organic C

Fixed

N

After Paerl, et al., 1989

Fixed

N

Consortial N2 fixation between cyanobacteria and heterotrophic bacteria

Cyanobacte

rium

Heterotrophic Bacteria

Org

anic

Mat

ter

(C,N

)

Fix

ed N

CO

2

PO

43-

Vita

min

s, C

hela

tors

, Met

als

& O

ther

Gro

wth

Fac

tors

After Steppe, et al., 1996

Conclusions - 1

• Distinct shifts with nutrient enrichment in F1, F4, U3.

• Oligotrophic periphyton has diverse diazotrophs, including cyanobacteria, -, - & -proteobacteria

• In eutrophic areas, diversity is limited, cyanobacterial species dominate, indicating a noticeable shift to bloom forming genera.

• Methanotrophs may be important groups of nitrogen fixers.

2. nifH expression in epiphyton over a diel period

Epiphyton ‘sweaters’

Genomic DNA

Nested PCR

Sequence analysis

Cloning and RFLP analysis

Total RNA

RT PCR

Epiphyton sample

Image source: www.dr-addie.com

0.1 substitutions/site

Frankia sp. CcI3

Phormidium sp. AD1 U3-30

U3-17

Calothrix sp.

U3-9

U3-37

U3-38

Uncultured clone BS0799(2130) R11

U3-7

Uncultured bacterium BS0797 D07

Nostoc sp.

U3-25

Pseudanabaena PCC7403

U3-23

U3-18

Anabaena 7120

Chlorogloeopsis sp.

U3-14

U3-10

Anabaena sp. I1

Marine stromatolite eubacteriumHB(089)

U3-13

U3-11

Uncultured bacterium clone GN1063A8

Dermocarpa sp.

U3-2

Uncultured bacterium clone MO163H23

Uncultured bacterium clone lg1114

Cyanothece sp. WH 8902

Uncultured bacterium clone Yushu-10

U3-16

Myxosarcina sp. Xenococcus sp.

U3-20 U3-8

Halothece sp. MPI 96P605 Uncultured bacterium clone GN2084A19

U3-35 Gloeothece sp. Marine stromatolite cyanobacterium HBCFischerella UTEX1931

U3-15 U3-3

Uncultured forest soil clone DUNnif298Chroococcidiopsis thermalis PCC 7203

U3-31 U3-21

Fischerella UTEX1931 FSU73140

Uncultured eubacterium Ice aggregateUncultured bacterium clone M1a-5

Nostoc PCC 6720 Anabaena variabilis

Plectonema boryanum Plectonema boryanum (IU 594)

Uncultured bacterium clone MING-36AUncultured bacterium clone BSC-2

U3-24 U3-19

U3-32 U3-28

U3-34 U3-29

U3-33 U3-12

U3-36 U3-5

Uncultured bacterium NR1633 Uncultured cyanobacterium clone NRE5

U3-26 U3-4

U3-27 U3-22

Lyngbya lagerheimii (UTEX 1930)Marine stromatolite eubacterium

U3-6 U3-1

Desulfovibrio vulgaris Spirochaeta aurantia

Chlorobium tepidum TLSChlorobium tepidum

Azotobacter chroococcum MCD 1Sinorhizobium sp. TJ170

100

93

99

50

100

83

77

100

92

5964

100

100

90

96

100

70

90

92

100

100

92

100

80

87

100

85

100

100

100

99

100

81

54

100

58

100

64

98

100

81

Rarefaction & phylogenetic analyses of nifH DNA clones

Cyanobacterial cluster

-Proteobacterial cluster

Distribution of clones

Epiphytic DNA based sequences

Cluster I (non heterocystous

unicellular cyanobacteria)

15%

Cluster VII (unidentified

cyanobacteria)9% Cluster VIII (-

proteobacteria)3%

Cluster VI (Uncultured

cyanobacterium clones - II)

25%

Cluster V (unidentified

cyanobacteria)10%

Cluster II (heterocystous cyanobacteria)

8%Cluster IV

(Uncultured cyanobacterium

clones - I)19%

Cluster III (non heterocystous cyanobacteria)

11%

Phylogenetic analysis of RT-PCR nifH clones

0.1 substitutions/site

Frankia sp. CcI3

Lyngbya lagerheimii (UTEX 1930) Uncultured bacterium clone GN1063A8

Marine stromatolite eubacterium HB(08919.15-9

Uncultured bacterium clone GN1063A3Uncultured bacterium clone GN1062A0

Calothrix sp. Uncultured bacterium cluster K

19.15-15

7.15-16

23.15-10

7.15-10

23.15-16 23.15-7

Anabaena sp. I1

Uncultured bacterium clone lg1117

Unidentified cyanobacterium

23.15-13

Fischerella UTEX1903

23.15-12

19.15-11

Gloeothece sp.

15.15-8

15.15-11

7.15-15

11.15-10

19.15-6

23.15-14

Uncultured bacterium clone Yushu-11

7.15-17

19.15-8

7.15-21

19.15-3

Sinorhizobium sp. TJ170

Pseudanabaena PCC7403

15.15-7

Cyanothece sp. WH 8904

15.15-9

Azotobacter chroococcum MCD 1

7.15-6

7.15-4

23.15-11

Nostoc muscorum

23.15-2

19.15-14

7.15-22

23.15-8

15.15-10

7.15-2

19.15-16

19.15-13

Halothece sp. MPI 96P605

Synechocystis sp. WH 003

19.15-12

Dermocarpa sp.

Nodularia sp. KAC 13

11.15-4

Nostoc sp. Nostoc commune (UTEX 584)

Uncultured bacterium cluster F Scytonema sp.

Anabaena sp. L-31 Anabaena sp.

Anabaena 7120 Anabaena sp. A2

Nostoc PCC 6720 Anabaena variabilis

7.15-14 15.15-2

23.15-6 23.15-3

Uncultured bacterium clone MING-36A11.15-14

Plectonema boryanum Plectonema boryanum (IU 594)

19.15-5 15.15-4

7.15-1 7.15-9 7.15-5 15.15-6

7.15-23 19.15-10

7.15-24 7.15-20

11.15-13 11.15-6

Uncultured bacterium NR1629 Uncultured bacterium NR1619

Phormidium sp. Phormidium sp. AD1

11.15-7 11.15-1 11.15-9

11.15-5 19.15-7 19.15-2

23.15-9 23.15-5

15.15-12 15.15-5

15.15-3 15.15-1

Myxosarcina sp.Xenococcus sp.

Cyanothece sp. WH 8902 Cyanothece ATCC51142

Synechocystis sp. WH 002 Crocosphaera watsonii WH 8501

Calothrix sp. Fischerella UTEX1931

Chroococcidiopsis sp. Chroococcidiopsis thermalis PCC 7203

7.15-12 7.15-3

Symploca sp. PCC 8002 Microcoleus chthonoplastes

Trichodesmium sp. Trichodesmium sp.

Uncultured bacterium NR1633 Uncultured cyanobacterium clone NRE11.15-3 11.15-2

7.15-18 7.15-11

7.15-19 19.15-4

23.15-4 23.15-1

7.15-13 7.15-8 7.15-7 19.15-1

23.15-15 11.15-8

11.15-12 11.15-11

Desulfovibrio vulgaris Spirochaeta aurantia clone 2

100

100

89

100

50

82

83

100

100

96

50

63

100

98

100

74

70

100

91

57

100

100

84

100

95

73

79

90

56

78

99

89

70

70

100

100

73

86

91

53

51

100

56

100

6296

94

96

100

100

58

100

61

76

100

100

62

92

90

82

64

99

100

76

81

96

77

100

100

100

57

66

51

100

Cluster I

Cluster II

Cluster III

Cluster IV

Uncultured clone cluster

Cluster V

Cluster VI

Cluster VII

Unidentified cluster I

0

5

10

15

20

25

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Unidentified cluster II

0

10

20

30

40

50

60

70

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Unidentified cluster III

0

10

20

30

40

50

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Unidentified cluster IV

02468

1012141618

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Unidentified cluster V

0

2

4

6

8

10

12

14

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Unidentified cluster VI

0

0.5

1

1.5

2

2.5

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Unidentified cluster VII

0

2

4

6

8

10

12

14

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Uncultured cyanobacterium clones cluster

0

5

10

15

20

25

30

35

40

11.15 AM 3.15 PM 7.15 PM 11.15 PM 7.15 AM

time

# of clones

Distribution of RT-PCR nifH clones

0

10

20

30

40

50

60

70

Time11.15AM

Time15.15PM

Time19.15PM

Time23.15PM

Time7.15 AM

Time points

# of clones

Unidentifiedcluster I

Unidentifiedcluster II

Unidentifiedcluster III

Unidentifiedcluster IV

Unculturedcyanobacteriumclones clusterUnidentifiedcluster V

Unidentifiedcluster VI

Unidentifiedcluster VII

Conclusions - 2

• Specific cyanobacterial groups express nitrogenase as a function of time.

• Unidentified clusters novel to oligotrophic Everglades epiphyton observed.

• Nitrogenase expression suggest nitrogen limitation.

• Presence of deep branching limits confidence in assigning diazotrophic groups.

3. Methanotrophic diversity along the nutrient gradient in

periphyton mats

• Capable of growth on methane as sole source of carbon and energy

• Divided into 2 major phylogenetic groups:- Type I ( proteobacteria)

- Type II ( proteobacteria)

• Methane oxidation - by methane monooxygenases (pMMO & sMMO)

• nifH characterization implicate methanotrophs as diazotrophs in periphyton.

• High methanogenesis in eutrophic sites

Methanotrophic bacteria

0.1 substitutions/site

Nitrosomonas europaea Methylobacter sp. LW12

F4-1 F4-8

Methylosinus sporium

Uncultured bacterium clone Zhenjiang

Methylocystis sp. SD5

Methylocaldum sp. E10_A

F4-6

F1-4 F1-3

Methylocystis sp. 62/12

F1-5

U3-2

Methylocaldum gracile

Methylocystis sp. KS33

Methylosinus trichosporium

F4-4

Unculture bacterium clone Maahas 5F4-3

F1-1

Methylocaldum sp. ML184 Methylocaldum sp. ML100

Methylocaldum tepidum Methylocaldum szegediense

Uncultured bacterium clone Maligaya 1Uncultured bacterium clone Maligaya 2

Methylococcus capsulatus Methanotroph FL-DIKO

F4-5 F1-2

U3-3 U3-1

F1-7 F1-6

F4-7 F4-2

Methylosinus sporium Methylosinus sp. PW1

Type II methanotroph AML-A3 Methylocystis sp. 42/22

100

97

100

100

99

71

100

99

100

81

97

100

66

73

100

98

95

69

87

100

95

100

57

92

95

99

Phylogenetic analysis of pmoA clones from F1, F4 & U3

Type I & X methanotrophs

Unidentified clades

Type II methanotrophs

Distribution of pmoA clones from F1, F4 & U3

Conclusions - 3

• Type I methanotrophs dominate in three sites, suggesting N limitation.

• F4 harbor a greater diversity including type X and type II methanotrophs.

• F1 and U3 not diverse, and U3 represents single dominated unidentified clade.

• Presence in periphyton mats indicate methane oxidation.

Summary

• nifH diversity differs along the nutrient gradient

• Nitrogenase expression characterized as a function of time

• Expression patterns complex, suggests regulated diel nitrogen fixation

• Unique uncharacterized clades of diazotrophs and methanotrophs identified

Future Applications

• Genome characterization of nitrogen fixers.

• Assessment of environmental regulatory mechanisms.

• Mapping the flow of N in periphyton.

Acknowledgements

• Dr. Andrew V. Ogram• Committee members:

»Dr. Sue Newman»Dr. Edward Phlips

• National Science Foundation• Lab members• My family

“…Restoring the Everglades is not rocket science or brain Surgery.

It’s much more complicated than that…”

- Don Boesch University of Maryland