Different Compositions of Intestinal Nitrogen Fixation
Different Compositions of Intestinal Nitrogen Fixation Genes among Phylogenetic Groups of Termites (Insecta, Isoptera), with Emphasis on Higher Termites (Termitidae) Akinori Yamada1, 2, Moriya Ohkuma1, 3, Tetsushi Inoue3, Satoko Noda4, Atsuko Sugimoto5, Toshiaki Kudo1, 3, 6 1. RIKEN, 2. Kyoto University, 3 JST/ICORP, 4. JST/PREST, 5. Hokkaido Hokkaido University, 6. Yokohama City University The gut symbiotic microbes of termites, especially wood/litterwood/litter-feeders, fix atmospheric nitrogen to compensate for the low nitrogen concentrations in their foods. Nitrogen fixation fixation has been found in the following termite families: Termopsidae (Tmp), Kalotermitidae (Klt), Rhinotermitidae (Rhn), and Termitidae (Tmt). We investigated the compositions of nifH genes by cultureculture-independent methods in eight wood/litterwood/litterspecies: Zootermopsis nevadensis (Tmp), Schedorhinotermes medioobscurus (Rhn), Coptotermes gestroi (Rhn), Microcerotermes sp. sp. (Tmt), M. crassus (Tmt), Globitermes sulphureus (Tmt), Nasutitermes dimorphus (Tmt), and N. bicolor (Tmt) (Figure 1) 1). For the higher termites Microcerotermes spp., we analyzed not only the PCR products from DNA, but also RT (reverse transcription)transcription)-PCR products from mRNA of the gut community in order to know real contributions to the activity. The functional nifH sequences obtained from the eight species were assigned to any of the known three nifH groups: Anaerobe, ProteoProteo-Cyano, and AnfAnf-Methano (Figure 2) 2). Phylogenetic analysis of the nifH sequences from the present and previous studies revealed that the Anaerobe group group was further categorized to the four clusters: Anaerobe Ia, Ib, II, and III (Figure 3) 3). The major groups and/or clusters of the sequences derived from Tmp, Klt, Rhn, and Tmt were AnfAnf-Methano, AnfAnf-Methano and Anaerobe III, Anaerobe III, and Anaerobe Ib and II, respectively (Figure 4) 4). Based on the analyses of the RTRT-PCR products in the present and previous studies, it is quite possible that nitrogenases encoded by the major nifH groups and/or clusters of each family are also mainly transcribed in their guts (Figure 5, 6) 6). Meanwhile, the common nifH groups and/or clusters, though usually minor, were shared between two closely related termite families (Figure 7) 7). These suggest gradual shifts of nifH genes along the evolution of termites.
Soil
-
Soil or Wood
n.t. or ●
Soil or Wood
- or ●
Microcerotermes sp spp p. Globitermes sulphureus Nasutitermes spp.
Amitermes-gp Nasutitermitinae
NT N
n.t.
Termes-gp
NdK01
Soil
Anaerobe IA Anaerobe IB Anaerobe II Anaerobe III Anaerobe Other Anf-Methano Proteo-Cyano
NmS01
-
Cubitermes-gp
+ +
Termitinae
MspN06-WL
-
Soil
Rhinotermitidae
GsS01
Fungus--growers Fungus
Apicotermitinae
+
+++
+
McSE17-WL
Termitidae (Higher termite)
Macrotermitinae
Termopsidae Kalotermitidae
CFN
●
+ ++ +
+
RsN-T KY
●*
Wood
Schedorhinotermes medioobscurus Coptotermes gestroi
Rhinotermitidae
+++
Figure 7. Compositions of nifH group or cluster in the guts of four termite families. nifH genes are divided into three groups (Anf-Methano, Proteo-Cyano, Anaerobe), and Anaerobe group is further divided into four clusters (Anaerobe IA, IB, II, and III). +++: > 40%, ++: 20-40%, +: < 20%
CgK01
Wood
Kalotermitidae
+
CDN
●
Rhinotermitidae Termitidae
++ + +
+
SspS01
N N--fixation
Wood
+++
NKN
Food
Kalotermitidae
+++
GFN
This study
Zootermopsis nevadensis
Termopsidae
Anf-Methano Anaerobe III Proteo-Cyano Anaerobe IA Anaerobe II Anaerobe IB Others
HSN
Termopsidae
nifH group or cluster
ZnH01
Phylogeny of termties
Difference in composition of nifH gene among termite families
* Ohkuma et al. al. (1999) AEM, - = not detected, n.t n.t.. = not tested
Figure 1. Phylogy, utilizing food, and N fixaiton activity of termites.
Proteoroteo-cyano group
Figure 4. Composition of each nifH group or cluster in phylogenetically diverse termites.
RT--PCR (RNA) RT
Termite: Termitinae Microcerotermes crassus
A-5h
N fixation activities of three samples
Anaerobe group
24
26
30
24
26
28
30 24
26
28
30
300
Number of PCR cycles
C2H 4 nmol/g/h
250
Anf-methano group
28
A-48h
B-5h
200 150
PCR (DNA)
100 50
n.d.
0
Pseudo nif-group
A-5h
B-5h
A-48h
By acetylene reduction assay
24
26
28
30
24
26
28
30
24
26
28
30
Number of PCR cycles
Figure 2. A large phylogenetic tree showing the relative positions of major nifH groups
Figure from Ohkuma et al. al. (1999) AEM
Figure 5. N fixation activity and expression of nifH. 78
87 59
Mc DEF-WL-D-05 Mc DEF-WL-D-18 Mc DEF-WL-D-08 Mc DEF-WL-D-02 Gs S-14 Mc N-WL-D-03a Mc N-WL-D-15 Mc N-WL-D-20a 89
70
97
77
NdK-05
SmS01 22 Mc N-WL-D-04 Mc N-WL-D-05 Mc DEF-WL-D-07 CfN12 NdK-22 SmS01 05
88
69
Mc N-WL-D-22 Gs S-15 Mc N-WL-D-01 Gs S-07 Gs S-13 Gs S-03 Mc N-WL-D-10 Mc N-WL-D-16 Mc DEF-WL-D-12 Mc DEF-WL-D-21 Mc DEF-WL-D-22 Mc N-WL-D-07 NTN36 NdK-01 NdK-12 NdK-10 NTN6 NspS-01a NTN18 66 NspS-14 Gs S-01 Gs S-18
Cluster
NspS-18 NTN21 NspS-06 NTN2 NspS-10
Anaerobe II:
CfN10 C.c ello bio parum
Termitidae
DNA mRNA
DNA mRNA
DNA mRNA Anaerobe IA Anaerobe IB Anaerobe II Anaerobe III Anaerobe Other Anf-Methano Proteo-Cyano
53
70 55
CdN7
TKY2
99
C.beije rinck ii
100
TKY9 HSN20 CgK-09 CgK-13 CfN5
74
TKY6 CgK-10 NKN20
67
NTN24
100 100 NKN5
91
TKY7
TKY15
100 NdK-07 NdK-23 ZnH01 30 ZnH01 31
100 CgK-16 57
100 SmS01 02
71
94 91
CfN16 100
75
63 74
64
CgK-19 CgK-07 CgK-01 CgK-02
CfN13 GFN14 GFN8 NKN6 CdN8 ZnH01 04 ZnH01 33 ZnH01 22 SmS01 11
64 76
51
100 C.acetobuty licum C.pas teurianum2 C.pas teurianum4 C.pas teurianum1 100 100
60
Rhinotermitidae
Anaerobe IB:
Termitidae
CfN24
Anaerobe III: 65
72
96
88
52
Anaerobe IA:
ZnH01 15
93
68 62 88
81
CgK-06 CfN22
CfN4 CfN21 CfN3
Gs S-21g NspS-03 NspS-15 NspS-12 NspS-11 NdK-04 NspS-13 Mc N-WL-D-09 NTN30
ZnH01 35
88
58
GFN25
CdN1 CdN3 99
0.05
GFN10
TKY3 TKY16
100 74
SmS01 14
MS.ac etiv oransC2A MS.bark eri MS.maz eiGoe1 100
99
82
ZnH01 19 ZnH01 29 TreponemaZAS-9 CdN28 SmS01 08 TreponemaZAS-2 ZnH01 07
S.z uelz erae D.gigas C.tepidum CgK-12 SmS01 03 SmS01 18
CdN14
A.v inelandii k .pneumonia e S.meliloti Anabaena
Termopsidae Kalotermitidae Rhinotermitidae
Figure 3. Four clusters in Anaerobe group. The major families of termites which compose each cluster are shown.
Termitidae (M. crassus)
Kalotermitidae (N. koshunensis*) Rhinotermitidae (C. formosanus**)
* Noda et al. (1999) AEM **Noda et al. (2002) M&E
Figure 6. Composition of each group or cluster of nifH for DNA and mRNA.
Soil
-
Soil or Wood
n.t. or ●
Soil or Wood
- or ●
Microcerotermes sp spp p. Globitermes sulphureus Nasutitermes spp.
Amitermes-gp Nasutitermitinae
NT N
n.t.
Termes-gp
NdK01
Soil
Anaerobe IA Anaerobe IB Anaerobe II Anaerobe III Anaerobe Other Anf-Methano Proteo-Cyano
NmS01
-
Cubitermes-gp
+ +
Termitinae
MspN06-WL
-
Soil
Rhinotermitidae
GsS01
Fungus--growers Fungus
Apicotermitinae
+
+++
+
McSE17-WL
Termitidae (Higher termite)
Macrotermitinae
Termopsidae Kalotermitidae
CFN
●
+ ++ +
+
RsN-T KY
●*
Wood
Schedorhinotermes medioobscurus Coptotermes gestroi
Rhinotermitidae
+++
Figure 7. Compositions of nifH group or cluster in the guts of four termite families. nifH genes are divided into three groups (Anf-Methano, Proteo-Cyano, Anaerobe), and Anaerobe group is further divided into four clusters (Anaerobe IA, IB, II, and III). +++: > 40%, ++: 20-40%, +: < 20%
CgK01
Wood
Kalotermitidae
+
CDN
●
Rhinotermitidae Termitidae
++ + +
+
SspS01
N N--fixation
Wood
+++
NKN
Food
Kalotermitidae
+++
GFN
This study
Zootermopsis nevadensis
Termopsidae
Anf-Methano Anaerobe III Proteo-Cyano Anaerobe IA Anaerobe II Anaerobe IB Others
HSN
Termopsidae
nifH group or cluster
ZnH01
Phylogeny of termties
Difference in composition of nifH gene among termite families
* Ohkuma et al. al. (1999) AEM, - = not detected, n.t n.t.. = not tested
Figure 1. Phylogy, utilizing food, and N fixaiton activity of termites.
Proteoroteo-cyano group
Figure 4. Composition of each nifH group or cluster in phylogenetically diverse termites.
RT--PCR (RNA) RT
Termite: Termitinae Microcerotermes crassus
A-5h
N fixation activities of three samples
Anaerobe group
24
26
30
24
26
28
30 24
26
28
30
300
Number of PCR cycles
C2H 4 nmol/g/h
250
Anf-methano group
28
A-48h
B-5h
200 150
PCR (DNA)
100 50
n.d.
0
Pseudo nif-group
A-5h
B-5h
A-48h
By acetylene reduction assay
24
26
28
30
24
26
28
30
24
26
28
30
Number of PCR cycles
Figure 2. A large phylogenetic tree showing the relative positions of major nifH groups
Figure from Ohkuma et al. al. (1999) AEM
Figure 5. N fixation activity and expression of nifH. 78
87 59
Mc DEF-WL-D-05 Mc DEF-WL-D-18 Mc DEF-WL-D-08 Mc DEF-WL-D-02 Gs S-14 Mc N-WL-D-03a Mc N-WL-D-15 Mc N-WL-D-20a 89
70
97
77
NdK-05
SmS01 22 Mc N-WL-D-04 Mc N-WL-D-05 Mc DEF-WL-D-07 CfN12 NdK-22 SmS01 05
88
69
Mc N-WL-D-22 Gs S-15 Mc N-WL-D-01 Gs S-07 Gs S-13 Gs S-03 Mc N-WL-D-10 Mc N-WL-D-16 Mc DEF-WL-D-12 Mc DEF-WL-D-21 Mc DEF-WL-D-22 Mc N-WL-D-07 NTN36 NdK-01 NdK-12 NdK-10 NTN6 NspS-01a NTN18 66 NspS-14 Gs S-01 Gs S-18
Cluster
NspS-18 NTN21 NspS-06 NTN2 NspS-10
Anaerobe II:
CfN10 C.c ello bio parum
Termitidae
DNA mRNA
DNA mRNA
DNA mRNA Anaerobe IA Anaerobe IB Anaerobe II Anaerobe III Anaerobe Other Anf-Methano Proteo-Cyano
53
70 55
CdN7
TKY2
99
C.beije rinck ii
100
TKY9 HSN20 CgK-09 CgK-13 CfN5
74
TKY6 CgK-10 NKN20
67
NTN24
100 100 NKN5
91
TKY7
TKY15
100 NdK-07 NdK-23 ZnH01 30 ZnH01 31
100 CgK-16 57
100 SmS01 02
71
94 91
CfN16 100
75
63 74
64
CgK-19 CgK-07 CgK-01 CgK-02
CfN13 GFN14 GFN8 NKN6 CdN8 ZnH01 04 ZnH01 33 ZnH01 22 SmS01 11
64 76
51
100 C.acetobuty licum C.pas teurianum2 C.pas teurianum4 C.pas teurianum1 100 100
60
Rhinotermitidae
Anaerobe IB:
Termitidae
CfN24
Anaerobe III: 65
72
96
88
52
Anaerobe IA:
ZnH01 15
93
68 62 88
81
CgK-06 CfN22
CfN4 CfN21 CfN3
Gs S-21g NspS-03 NspS-15 NspS-12 NspS-11 NdK-04 NspS-13 Mc N-WL-D-09 NTN30
ZnH01 35
88
58
GFN25
CdN1 CdN3 99
0.05
GFN10
TKY3 TKY16
100 74
SmS01 14
MS.ac etiv oransC2A MS.bark eri MS.maz eiGoe1 100
99
82
ZnH01 19 ZnH01 29 TreponemaZAS-9 CdN28 SmS01 08 TreponemaZAS-2 ZnH01 07
S.z uelz erae D.gigas C.tepidum CgK-12 SmS01 03 SmS01 18
CdN14
A.v inelandii k .pneumonia e S.meliloti Anabaena
Termopsidae Kalotermitidae Rhinotermitidae
Figure 3. Four clusters in Anaerobe group. The major families of termites which compose each cluster are shown.
Termitidae (M. crassus)
Kalotermitidae (N. koshunensis*) Rhinotermitidae (C. formosanus**)
* Noda et al. (1999) AEM **Noda et al. (2002) M&E
Figure 6. Composition of each group or cluster of nifH for DNA and mRNA.
