| A systematic screening for agar-degrading bacteria from the seawater samples yielded 87 isolates with promising agarolytic activity, among which 15 strains were selected for further analysis. To investigate the phylogenetic position of these strains,the 16S rDNA sequences were cloned, sequenced and compared with those of related strains. A molecular phylogenetic dendrogram was constructed based on genetic distance analysis.Sequence comparison allowed to classify the strains as members of genera Cellulophaga, Cytophaga, Microbulbifer, Glaciecola, Pseudoalteromonas, Pseudomonas, Alteromonas and Agarivorans. Strains QM5, QM21 and QM36 were identified to be Cellulophaga lytica. Strains QM15 and QM28 were identified to be Glaciecola mesophila. Strains QM11, QM35, QM47 and QM65 were identified to be Cytophaga fucicola, Pseudoalteromonas atlantica, Pseudoalteromonas haloplanktis and Alteromonas addita, respectively.Two strains, QM42 and DH166, were isolated from coastal seawater of Qingdao and the East China Sea, respectively. Both of them are gram negative marine bacteria. They can't be cultured without NaCl supplement. Strain QM42 was positive in tests of catalase, oxidase, aminopeptidase and urease, but negative in the test of Voges Proskauer. This organism could grow and ferment glucose, xylose, lactose, and cellobiose. Strain DH166 was positive in tests of catalase, oxidase, gelatinase and lecithinase, but negative in the test of Voges Proskauer. This organism could not be cultured with 1% and 6% NaCl supplement. The major fatty acids detected in strain QM42 were 16:1w7c/15 iso 2-OH (29.11 %), 16:0 (19.53%), 18:1 w7c (19.62%), 18:0(10.78%), 12:0(7.56%), 12:0 2OH(2.31%), 12:0 3OH(3.27%), 10:0 3OH(3.53%), 14:0(1.89%) and 17:0(1.09%). The major fatty acids detected in strain DH166 were 16:1w7c/15 iso 2-OH (11.57 %), 16:0 (9.26%), 18:1 w7c (4.27%), 15:0(10.77%), 12:0 3OH(2.26%), 11:0 3OH (6.60%), 15:1 w8c (13.69%), 17:1 w8c (21.19%), 17:1 w6c (1.94%) and 17:0 (8.61%). Comparative 16S rRNA gene sequence analysis revealed that strain DH166 shared approximately 95.8% sequence similarity with members of the genus Pseudoalteromonas. But the partial sequences of the 16SrDNA of QM42 only showed similarities of about 90% to different members of the genus Microbulbifer. Further phylogenetic analysis of the 16S rRNA gene sequence showed that strain DH166 formed a separate branch within the genera Pseudoalteromonas. Thus strain DH166 maybe represent a potential novel species in the genera.The strain QM42 could be differentiated from its closest phylogenetic neighbours on the basis of several phenotypic features. It probably be a member of a new species and may be a member of a new genus in theγ-proteobacteria.The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. This strain was gram negative, obligately aerobic, curved rod and polarly flagellated. On the basis of several phenotypic characters, biochemical and morphological characters and a phylogenetic analysis of the gene coding for the 16S rRNA, this strain was identified as Agarivorans albus strain QM38. An extracellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40℃, pH 7.6. Its activity was greatly affected by different concentration of agarose. The hydrolysis products were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE).Threeβ-agarase genes, agaD01, agaD02 and agaD03, were amplified by PCR using chromosomal DNA of strain QM38 as a template. Two of them, agaD02 and agaD03, were first described in genus Agarivorans. The accession numbers for agaD02 and agaD03 in GenBank were EF051475 and EF199908, respectively. Homology searches were carried out with a BLAST program at the National Center for Biotechnology Information (NCBI) website. The nucleotide sequences of the agaD01, agaD02 and agaD03 gene were compared with entries in the GenBank database. The agaD01 gene were 97.8% and 98.9% identical to the two agarase genes from vibrio sp. JT0107 and Agarivorans sp. JA-1, and 98.2% with agaA11 gene from agarivorans sp. JAMB-A11. The agaD02 gene showed 98.8% sequence identify to the agaB gene from vibrio sp. JT0107. The agaD03 gene had a identify of 96.8% and 96.5% with two agarase genes from vibrio sp. PO-303 and Pseudoalteromonas sp.CY24. The deduced amino acid sequences of the agaD01 and agaD02 genes were compared with entries in the DDBJ database. The proteins sequences were analyzed by molecular software tools.According to the sequence of agaD02 gene, a pair of primers were respectively designed and synthesized. After amplification with PCR, the product was cloned into pMD19-T simple vector using TA cloning. The recombinants were sequenced and identified by restrictive endonuclease digestion. The target sequences were then subcloned into a highly efficient eukaryotic expression vector pET24a. The expression vectors were identified by double restrictive endonuclease digestion and then were transformed into E. coli BL21(DE3).The recombinant E. coli BL21 (pET24a-agaD02) were induced by IPTG on Petri dish and were dyed with Lugol iodide solution after culture for 24h at 37℃. A clear Zone was observed around the colony of the E. coli. This is to certificate that the pET24a-agaD02 eukaryotic expression vectors are successfully constructed.An extracellularβ-agarase was purified to electrophoretic homogeneity by ammonium sulfate precipitation and successive liquid chromatographies. The molecular mass of the enzyme was determined to be 44.2 kDa by SDS-PAGE. Thisβ-agarase might be encoded by the agaD03 gene.
|
PDF Full Text Request