Distribution and Phylogenetic Diversity of CbbL Gene Encoding

RuBisCo in the Deep-sea Sediments from the South China Sea

Jie Su

, Hongxia Ming

, Quanrui Chen

1,2

, Yuan Jin

1,3

, Chunxin Zhang

1,2

, Daoming Guan

and

Jingfeng Fan

1,3*

Key Laboratory for Ecological Environmental in Coastal Areas(SOA), National Marine Environmental Monitoring

Center, Dalian China 116023;

College of Fisheries and Life Science, Dalian Ocean University, Dalian China116023;

Fourth Institute of Oceanography, State Oceanic Administration, Beihai China 536000

Email: jffan@nmemc.org.cn

Keywords: Phylogenetic diversity, deep-sea sediments, South China Sea

Abstract: RuBisCO is the key enzyme catalyzing the first and major step of carbon fixation in Calvin cycle. With the

aim to examine the phylogenetic diversity of RuBisCO genes, cbbL gene was amplified by PCR from three

South China Sea deep-sea sample, cloned, and sequenced. A total of 28 OTUs deriving from 236 cbbL

clones covered 16 phylotypes which all belong to Proteobacteria. The estimated coverage values showed

that more than 85% of bacterial cbbL diversity was captured. Shannon and Simpson indices indicated a high

diversity of the total cbbL gene in the SCS deep-sea sediments. In conclusion, microbial RuBisCO genes in

the South China Sea display a broad range of phylogenetic diversity. The predominant group in these three

deep-sea sediments included Thiobacillus and Thiorhodococcus, which was chemoautotrophic bacteria

involving in Calvin–Benson–Bassham cycle. The above results implicate that bacteria with the potential for

carbon dioxide fixation and chemoautotrophy oocur in the South China Sea.

1 INTRODUCTION

is the major contributor to global warming and

reduction of CO

input in atmosphere is essential for

control global warming. The ocean is recognized as

a huge carbon reservoir. Deep-sea sediments are a

huge carbon pool, the study of microbiology in

deep-sea ecosystem mediating flows of energy in

metabolism will help understand the CO

fixation

capacity of the marine ecosystem (Coffin, 2004).

Biologically mediated CO

fixation is a major

pathway in marine ecosystem. Microbiological

auotrophic CO

fixation is widely distributed and

adapted to many habitats.

Autotrophic CO

fixing bacteria do not belong to

a specific taxonomic group and occurs in many

species. Most chemolithoautotrophic bacteria

mediate autotrophic CO

fixation via the Calvin–

Benson–Bassham cycle (Shively, et al., 1986; Selesi

et al., 2005) RuBisCO is the key enzyme catalyzing

the first and major step of carbon fixation in the

Calvin cycle and exists in multiple natural forms

which differ in structure, catalytic property, and O2

sensitivity. As such, RuBisCO form I-encoding

cbbL genes have been used as functional markers for

molecular ecological studies of CO

assimilative

autotrophs in aquatic systems (Yuan et al., 2012;

Kovaleva et al., 2011).

The South China Sea (SCS), near to the West

Pacific “warm pool” is the biggest and deepest sea

in China (Dai et al, 2002). SCS may be enriched in

fixation bacteria. However, there are no report

on the cbbL gene diversity from the SCS at the

moment.

In this study, the geographical distribution and

phylogenetic diversity of cbbL gene in sediments of

the South China Sea were determined with the aim

to broaden our view on the diversity of different

deep-sea habitat.

Su, J., Ming, H., Chen, Q., Jin, Y., Zhang, C., Guan, D. and Fan, J.

Distribution and Phylogenetic Diversity of CbbL Gene Encoding RuBisCo in the Deep-sea Sediments from the South China Sea.

In Proceedings of the International Workshop on Environment and Geoscience (IWEG 2018), pages 5-10

ISBN: 978-989-758-342-1

2 MATERIAL AND METHODS

2.1 Sample Collection

Deep-sea surface sediments (5 cm lower than the top

layer) were collected using a gravity box-coring

device from three sampling site in the Nansha area

of the South China Sea in the Spring of 2013.

Characteristics of three sampling sites were

described in Table 1.

Table 1: Sample information.

Sites Water

Depth(m)

pH eh

(mv)

Temperature Sample

describe

NSCA 1743 7.44 172 2.9 Yellow

mud

NSCE 1683 7.35 176 3.0 Brown

mud

NSCI 963 7.37 152 11.1 Red mud

2.2 DNA Extraction

Total genomic DNA was extracted from 5 g

sediments following with the Rapid Soil DNA

Isolation Kit (Sangon Biotech, Shanghai, China) and

further purified using the QIAquikPCR purification

kit according to the manufacturer’s directions.

2.3 Amplification, Cloning and

Sequencing of CbbL Gene

The purified DNA was used for cbbL gene

amplification using primers 595f and 1387r (Hung et

al, 2012). Thermo cycling reaction was as

followed:95℃ annealing for 5min;35 cycles of 94℃

for 30 s,52℃for 30 s,72℃for 1min;72℃for

10min.The PCR products was cloned using the

TOPO TA cloning kit (Invitrogen, Carlsbad, CA,

USA) and Escherichia coli TOP10 competent cells.

The inserted cbbL gene was sequenced by a

commercial company (Takara, Dalian).

2.4 Phylogenetic Analysis

Preliminary analysis of the sequences was

performed using BLAST (http://www.

ncbi.nlm.nih.gov/blast/). The nucleotide and inferred

amino acid sequences were aligned with sequences

from GenBank using CLUSTAL W (Thompson et

al., 1994). The aligned sequence with more than 97%

similarity was defined as one Operational

Taxonomic Unit (OTU). Phylogenetic tree of

bacteria harbouring cbbL gene was constructed

using neighbour-joining algorithm within the

MEGA 5.0 software. Alpha-diversity indices,

including Shannon, and Simpson values, were

subsequently calculated by Mothur.

3 RESULTS

3.1 Diversity Analysis of Cbbl Clone

Libraries

Bacterial cbbL sequences were obtained from all

three samples (NSCA, NSCE, NSCI). A total of 236

clones were achieved from three cbbL clone

libraries constructed from the SCS deep-sea

sediments. When analyzed using a 97% sequence

similarity cut oﬀ, the 236 total cbbL sequences

formed 28 OTUs. Rarefaction analysis of the clone

libraries showed that the species accumulation

curves were asymptotic for all three libraries (Figure

1), indicating a good coverage of the total cbbL gene

diversity in the SCS deep-sea sediments. The

estimated coverage values showed that more than

85% of bacterial cbbL diversity was captured (Table

2). This result indicates the capacity of cbbL clone

libraries is large enough for diversity analysis.

Shannon and Simpson indices were calculated to

evaluate the evenness and diversity of the cbbL at

each site. Shannon-Wiener index of cbbL clone

libraries from high to low is NSCE, NSCI and

NSCA respectively, suggesting the NSCE site

sediments is in the highest bacterial diversity among

the three sampling sites.

Figure 1: Rarefaction curves for the cbbL.

IWEG 2018 - International Workshop on Environment and Geoscience

Table 2: Diversity indices for the cbbL clone libraries.

Clone library No. of clones No. of OTUs

Coverage (%)

Shannon-Wiener

index

Simpson index

NACS 76 8 89.4 2.03 0.86

NACE 80 11 86.2 2.2 0.88

NSCI 80 9 88.7 2.1 0.87

Percentage of coverage is the percentage of observed number of OTUs divided by the Chao1 estimated value

For the Shannon diversity index, a higher number represents greater diversity

For the Simpson diversity index, a higher number represents greater diversity

Figure 2: Phylogenetic analysis of bacteria harbouring cbbL gene derived from sediments of NSCA site.(a)

Phylogenetic tree based on the cbbL (translated amino acids) sequences;(b)Distribution of cbbL gene in deep-sea

sediments.

3.2 Phylogenetic Analysis of Cbbl

Genotypes

The expected size fragment was amplified with

DNA extracted from three sediment samples and

preliminary analysis of sequence yielded positive

results with the cbbL gene. A total of 28 OTUs from

three cbbL clone libraries covered 16 phylotypes:

Thiobacillus, Thiorhodococcus, Thiomonas,

Halothiobacillus, Halorhodospira halochloris,

Acidithiobacillus, Ectothiorhodospira, Thiobacillus

denitrificans, Nitrosomonas, Hydrogenophaga,

Thialkalivibrio denitrificans, Thiocys,

Thialkalivibrio, Thiobacillus denitrificans,

Chromatium and Rhodobacter. It may be concluded

that all of the cbbL sequences detected in Deep-sea

sediments belong to the Proteobacteria. The

phylogenetic analysis results were matching with the

above diversity indices, indicating the diversity of

the cbbL gene in the South China Sea.

The bacterial communities harbouring cbbL gene

derived from NSCA site were clustered into three

classes: α-Proteobacteria, β-Proteobacteria and γ-

Proteobacteria; 8 genera: Thiobacillus,

Thiorhodococcus, Thiocystis, Thialkalivibrio,

Ectothiorhodospira, Proteobacteria, Chromatium

and Rhodobacter (Figure 2). The predominant group

in NSCA site deep-sea sediments included

Thiobacillus, Thiorhodococcus, Thiocystis,

Thialkalivibrio, which was chemoautotrophic

bacteria involving in Calvin–Benson–Bassham cycle.

Distribution and Phylogenetic Diversity of CbbL Gene Encoding RuBisCo in the Deep-sea Sediments from the South China Sea

Figure 3: Phylogenetic analysis of bacteria harbouring cbbL gene derived from sediments of NSCE site.(a) Phylogenetic

tree based on the cbbL (translated amino acids) sequences;(b)Distribution of cbbL gene in deep-sea

sediments.

Figure 4: Phylogenetic analysis of bacteria harbouring cbbL gene derived from sediments of NSCI site.(a) Phylogenetic

tree based on the cbbL (translated amino acids) sequences;(b)Distribution of cbbL gene in deep-sea sediments.

IWEG 2018 - International Workshop on Environment and Geoscience

The bacterial communities harbouring cbbL gene

derived from NSCE site were clustered into two

classes:β-Proteobacteria and γ-Proteobacteria; 11

genera: Thiobacillus, Thiorhodococcus,

Halorhodospira halochloris, Thialkalivibrio

denitrificans, Ectothiorhodospira, Thiomonas,

Thiobacillus denitrificans, Nitrosomonas,

Hydrogenophaga, Thiobacillus denitrificans and

Thioalkalivibrio (Figure 3). The major bacterial

groups in the NSCE site sediments was found to

include Thiobacillus, Thiorhodococcus,

Halorhodospira halochloris, Thialkalivibrio

denitrificans, Ectothiorhodospira, also with low

abundant of Nitrosomonas, Hydrogenophaga,

Thiobacillus denitrificans and Thioalkalivibrio.

In the phylogenetic tree constructed from the

phylotypes of NSCE clone libraries, eleven OTUs

could be assigned to three classes: β-Proteobacteria,

γ-Proteobacteria and Acidithiobacillia; 9 genera:

Thiobacillus, Thiorhodococcus, Thiomonas,

Halothiobacillus, Acidithiobacillus,

Ectothiorhodospira, Thiobacillus denitrificans,

Nitrosomonas and Hydrogenophaga (Fig. 4).

Thiobacillus was the most dominant group and

accounted for 20% of in NSCI site deep-sea

sediments. Other predominant genera in NSCA site

deep-sea sediments included Thiorhodococcus and

Thiomonas, which were also chemoautotrophic

bacteria involving in Calvin–Benson–Bassham

cycle.

4 DISCUSSIONS

The RuBisCO gene were detectable in the SCS

deep-sea sediments and the general richness of the

cbbL gene was relatively high (from 0.11to 0.14

OTU per clone). Similar results were reported from

other habitats. The richness of cbbL genes Giri et al.

detected (0.12 OTU per clone) in Mono Lake was

comparable to the richness we observed, despite the

differences in habitat diversity (Giri et al., 2004).

Elsaied et al. identified the richness of cbbL genes

(0.10 OTU per clone) covering a range of habitats

associated with a hydrothermal vent site, including

sediment, overlying water, and as symbionts

(Elsaied and Naganuma, 2001). However, RuBisCO

genes with low richness were also observed in some

extreme habitats such as the deep-sea hydrothermal

vents, volcanic deposits and deep hypersaline anoxic

basin (Elsaied and Naganuma, 2001; Nanba et al,

2004; Elsaied et al, 2007; Wielen, 2006). Therefore,

the diversity of the cbbL gene may be correlated

with certain characteristics of the microbial habitats.

The amplicons of the cbbL gene all belonged to

form IA RuBisCO. This form is mainly found in

Alpha-, Beta- and Gammaproteobacteria, although a

few cyanobacterial sequences possess form IA as

well (Wielen, 2006). This study also indicated a

domination of the Proteobacteria distributed

throughout the SCS deep-sea sediments. Giri et al.

reported the similar results that the genus

Thiobacillus and Thiorhodococcus were the

dominant bacteria isolated from Mono Lake.

Thiobacillus-related RuBisCO were found to be

distributed globally and contribute to primary

production in the deep sea (Elsaied and Naganuma,

2001).Thiocystis with high proportion was detected

in NSCA site, while not detectable in NSCE and

NSCI. Rhodobacter as one genera of α-

proteobacteria was only present at the NSCA site,

also not detected in other two sites. The diversity of

bacterial populations in marine sediments maybe

due to the environmental characteristics difference

even in the same habitat. Among the detected groups

of the Gammaproteobacteria, the genera

Thioalkalivibrio were chemotrophic genus,

Halorhodospira and Ectothiorhodospira were

phototrophic genus.The 16 phylotypes that we

obtained from three SCS deep-sea sediments belong

to autotrophic bacteria and most were

chemoautotrophic bacteria. This was expected as

sampling site is located at 1000 m in the deep sea, a

depth at which light does not penetrate. Most of the

cbbL sequences detected in deep-sea sediments were

found belong to sulfur-oxidizing

Gammaproteobacteria and confirm the importance

of sulfur cycle bacteria in deep sea ecosystem.

Chromatium, Hydrogenophaga and

Ectothiorhodospira detected in this study were

facultative autotrophic bacteria. In conclusion, we

propose that the distribution of the deep-sea

RuBisCO genes cbbL may correlate with certain

characteristics of the microbial habitats.

ACKNOWLEDGEMENT

This work was supported by the National Key

Research Program (Grant 2016YFA0601400), the

State Oceanic Administration (Grant GASI-03-01-

02-05) of China and Key Laboratory for Ecological

Environmental in Coastal Areas (Grant 201813).

Distribution and Phylogenetic Diversity of CbbL Gene Encoding RuBisCo in the Deep-sea Sediments from the South China Sea

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