Chinese mitten crab (Eriocheir sinensis) is of great commercial significance and is bred extensively in Eastern and Northern China. Aquaculture is a complex ecosystem where microorganisms in the water, sediment and gastrointestinal tracts interact with each other to affect the health of aquatic animals (Cheng, Zhou, Xie, Ge, Zhu & Liu 2010). The microbial communities in crab pond water (Cheng et al. 2010) and in the gut of Chinese mitten crab (Li, Guan, Wei, Liu, Xu, Zhao & Zhang 2006) have been studied. However, there is no report on the microbial community in crab pond sediment.
Generally a crab may require 12–16 moults to reach maturity (Zhang, Li & Cui 2001). Crab shell is rich in chitin, and the crab pond represents one of the most chitin-rich environments. Organisms produce chitinase for different physiological purposes. Bacterial chitinases play roles in nutrition and parasitism, and thus form one of the major sources of chitinases (Dahiya, Tewari & Hoondal 2006). Chitinase genes have been identified in a large pool of uncultured chitinolytic microorganisms in marine and soil environments and used as molecular markers (Cottrell, Wood, Yu & Kirchman 2000; Metcalfe, Krsek, Gooday, Prosser & Wellington 2002). Recently, chitinases of various aquatic habitats have been investigated (LeCleir, Buchan & Hollibaugh 2004; Hobel, Marteinsson, Hreggvidsson & Kristjánsson 2005; Bhattacharya, Nagpure & Gupta 2007).
Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments generated by PCR is a reliable, rapid and an easy method to evaluate microbial diversity (Liu, Zhou, Yao, Shi, He, Benjamisen Hølvold & Ringø 2008; Zhou, Liu, Shi, He, Yao & Ringø 2009) and is capable to detect the dominant bacterial species in the environments (Muyzer, Waal & Uitterlinden 1993). We used PCR-DGGE and PCR screening technologies to investigate the microbial community in crab pond sediments.
The sediment sample was collected from a pond rearing Chinese mitten crab near the city of Nanjing, Jiangsu province, China, in July 2008. The depth of the pond was 1.5 m, with temperature of 30°C, pH 7.5 and the culture area was approximately 100 acres. The crabs were fed the commercial diet (Crude protein 36% and crude lipid 6% without dietary antibiotics) manufactured by Jiangsu Danyang Lekaihuai Feed Co., Ltd (Zhenjiang City, Jiangsu Province, China). The earth pond has been used for the crab culture for about 3 years. The pH of the water 50 cm below the surface was measured at 11:00 hours on the sampling day. Due that dissolved oxygen level in the crab pond water was above 5.0 mg L−1 and the surface of the pond sediment was sampled, the sediment sampled was not regarded to be anoxic.
The total genomic DNA of the crab pond sediment was extracted as described by Brady (2007) with some modifications. 10 g of sediment was suspended in a 50 mL centrifuge tube containing 20 mL of pre-heated (70°C) lysis buffer [100 mM Tris-HCl, 100 mM EDTA, 1.5 M NaCl, 1% (w/v) cetyl trimethyl ammonium bromide, 2% (w/v) SDS, pH 8.0], inverted to mix, and incubated in a 70°C water bath for 2 h with gentle inversion every 30 min. The tube was thereafter cooled down to 4°C on ice, and centrifuged at 10 000 g for 10 min at 4°C. The supernatant was transferred into a 50-mL centrifuge tube, and 20 mL of isopropanol was added for DNA precipitation. The genomic DNA was subjected to sequential digestion with RNase and proteinase K, and purified using a Cycle-Pure DNA kit (Omega, Norcross, GA, USA). The presence and size of purified DNA were visualized on an agarose gel using ethidium bromide staining.
Using the purified genomic DNA of sediment as the template, the variable V3-region on 16S rDNA was amplified with a pair of universal primers BA338f (5′-ACTCCTACGGGAGGCAGCAG-3′) including a 40-base GC clamp at the 5′ end and UN518r (5′-ATTACCGCGGCTGCTGG-3′). The PCR reaction and DGGE analysis were performed as described by Liu et al. (2008).
Using the method of LeCleir et al. (2004) that has successfully amplified the chitinase gene fragments of family 18 from different environmental samples, a degenerate primer set (GHI-F: 5′-GCGACGGIATGGAYYTIGAI-3′ and GHI-R: 5′-GGACGCCIGTCCAICC-3′) was designed based on the two conserved motifs, DGMDLD and GWTGV of family 18 chitinases (http://www.cazy.org/GH18.html).
A touchdown PCR was performed with the purified genomic DNA of the sediment as the template. The 50-μL PCR reaction system contained ~50- to 100-ng template DNA, 2.5 U Taq polymerase, 0.2 mM deoxynucleoside triphosphate, 1 mM of each primer and 2 mM Mg2+ in the buffer supplied by New England Biolabs. The PCR conditions were as follows: 5 min at 94°C, followed by five cycles of 94°C for 30 s, 60°C (decreasing by 1°C after each cycle) for 30 s and 72°C for 30 s, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 30 s, and a final extension at 72°C for 5 min. The presence and size of the PCR products were identified by agarose gels.
PCR products of the appropriate size (~450 bp) were retrieved, cloned into the vector pGEM-T Easy (Promega), and transformed into Escherichia coli DH5 (TaKaRa) using electroporation. One hundred transformants (white colonies) were randomly picked, subjected to PCR amplification with primers pGEM-F (5′-CCGACGTCGCATGCTCC-3′) and pGEM-R (5′-CTCCCATATGGTCGACCTG-3′), and sequenced by Sunbiotech (China). Nucleotide sequences of chitinase gene fragments were corrected by hand and translated into amino acid sequences by emboss Transeq (http://www.ebi.ac.uk/emboss/transeq). Alignment with other known sequences in the GenBank database was carried out by ncbi blast tool.
The representative nucleotide sequences of the chitinase gene fragments were deposited in the GenBank database under accession numbers GQ202084–GQ202091. All nucleotide sequences retrieved from the DGGE gel were deposited in the same database at HM756290–HM756301.
The sediment samples gave clear PCR products after amplification with primers BA338f and UN518r specific to V3 region of bacterial 16S rRNA gene sequences. The DGGE fingerprints from three replicates of the samples were similar (Fig. 1). To determine the taxon of predominant microbes, 12 distinct DGGE bands were excised and characterized by sequencing. These sequences were compared with that in the NCBI database (Table 1), and showed high identities (93–100%) with α-, β-, γ- and δ-Proteobacteria (six bands), Bacteroidetes (three bands) and two unclassified bacteria. Both PCR-DGGE and DGGE suggested that the dominant bacteria in crab pond sediment belonged to families Proteobacteria, Bacteroidetes and unclassified bacteria.
|Phylogenetic group||Band no.||Accession no.||Closest relative (obtained from blast search)||Identity (%)|
|α-Proteobacteria||11||HM756300||Uncultured bacterium clone MNO302A4 (GU996484)||93|
|β-Proteobacteria||6||HM756295||Uncultured Burkholderiales clone A6-48 (AM940566)||100|
|9||HM756298||Uncultured β-Proteobacterium clone MS026A1_C01 (EF701004)||100|
|γ-Proteobacteria||4||HM756293||Shigella flexneri strain FB5 (HQ701686)||99|
|7||HM756296||Uncultured γ-Proteobacterium clone OuchyA-10 (FN679150)||99|
|δ-Proteobacteria||2||HM756291||Uncultured Desulfobacteraceae bacterium (FN679112)||99|
|10||HM756299||Uncultured δ-Proteobacterium TH_c24 (GU998929)||98|
|Bacteroidetes||5||HM756294||Uncultured Flexibacteraceae clone HS-S-213 (HM592614)||98|
|8||HM756297||Uncultured Flexibacteraceae clone HS-S-164 (HM592609)||100|
|12||HM756301||Flavobacteriaceae clone CL101 (HQ686271)||99|
|Unclassified bacteria||1||HM756290||Uncultured bacterium clone 3H-233 (EU786128)||97|
|3||HM756292||Uncultured Chloroflexi clone CH-33 (AB293398)||98|
The DGGE results indicated that the dominant bacteria in the crab pond sediment were Proteobacteria, Bacteroidetes and unclassified bacteria. Proteobacteria also has been indicated to be dominant in crab pond water (Cheng et al. 2010), and Proteobacteria and Bacteroidetes are predominant in the gut of Chinese mitten crab (Li et al. 2006). Therefore, as Proteobacteria and Bacteroidetes dominant in crab pond sediment (Table 1), the result implied that the microbes in sediment may impact the microbial community structure in the gut of Chinese mitten crab.
Of the 100 randomly sequenced clones, 79 were identified to be putative chitinase gene fragments by BlastX searches. At amino acid level, these sequences showed 42–86% identities to known chitinases of various microbial sources, such as Actinobacteridae, Bacteroidetes, β-Proteobacteria and γ-Proteobacteria. After removing the redundant sequences using cd-hit program (Li & Godzik 2006), seven sequences showed divergence (sharing <95% identity) (Table 2). The length of these sequences varied with the range from 138 to 151 residues. Abundance analysis using distance-based operational taxonomic unit (out) and richness determination (dotur) software (Schloss & Handelsman 2005) showed that TDChi2 was the predominant out that represented 35 sequences. The amino acid sequences of these seven representative chitinase fragments were compared with other bacterial chitinases using the ClustalW program. The putative catalytic site residues, including a well-conserved region (DIDWE), are very similar to the conserved active site sequence (DXDXE) of family 18 chitinases (Synstad, Gåseidnes, van Aalten, Vriend, Nielsen & Eijsink 2004).
|Phylogenetic group||Sequence no.||Accession no.||Protein size (AA)||No. sequences||Microbial source||Relative identity (%)|
|Actinobacteridae||TDChi3||GQ202086||138||6||Streptosporangium roseum DSM 43021 (ACZ89829)||86|
|Bacteroidetes||TDChi2||GQ202085||144||35||Rhodothermus marinus DSM 4252 (AAU11838)||55|
|TDChi4||GQ202087||144||1||Salinibacter ruber DSM 13855 (ABC44395)||49|
|β-Proteobacteria||TDChi1||GQ202084||140||30||Chromobacterium sp. C-61 (AAP88583)||47|
|TDChi6||GQ202089||143||4||Burkholderia ambifaria MEX-5 (EDT39668)||42|
|γ-Proteobacteria||TDChi5||GQ202088||143||2||Stenotrophomonas sp. SKA14 (EED40275)||52|
|TDChi7||GQ202090||151||1||Stenotrophomonas maltophilia (AAB70917)||48|
Glycosyl hydrolases of family 18 have a conserved residue essential for catalytic activity (Papanikolau, Prag, Tavlas, Vorgias, Oppenheim & Petratos 2001). All the chitinase sequences identified in this study contain the conserved motif DXDXE, which can form a (βα)8 barrel (TIM barrel) structure (Synstad et al. 2004; Vaaje-Kolstad, Vasella, Peter, Netter, Houston, Westereng, Synstad, Eijsink & Aalten 2004). In addition, the sequences obtained in the present study showed 42% to 86% similarities to known sequences from Actinobacteridae, Bacteroidetes and Proteobacteria (Table 2), suggesting that the predominant microbes in crab pond sediment are mainly chitinase-producing bacteria. It has reported Actinobacteria have an important chitinolytic function in soil and vermicompost ecosystems (Metcalfe et al. 2002; Yasir, Aslam, Kim, Lee, Jeon & Chung 2009). Chitinase gene fragments have been cloned and sequenced from cultivated Actinobacteria from lake sediments (Xiao, Yin, Lin, Sun, You, Wang & Wang 2005). However, few Bacteroidetes chitinase sequences in soil and vermicompost ecosystems have been reported. It is suggested that chitinoclastic communities in different environment have specificity to some extent.
Chitinases are widely distributed among the members of diverse Proteobacteria lineages (Cottrell et al. 2000) and Actinobacteria (Xiao et al. 2005). The result is in agreement with earlier studies that Proteobacteria are one of the dominant species attached to the chitinous outer layer of setae (Goffredi, Jones, Erhlich, Springer & Vrijenhoek 2008). Furthermore, many marine bacteria, particularly chitin-degrading Vibrios possess complex signal transduction systems for finding and adhering to chitinous substrates (Yu, Bassler & Roseman 1993; Keyhani & Roseman 1999). Based on this information we put forward the hypothesis that this is the main reason why Proteobacteria is dominant bacteria in water, sediment and gut. Based on the results we suggest to investigate novel chitinase genes in these environments.