Chryseobacterium chengduensis sp. nov. isolated from the air of captive giant panda enclosures in Chengdu, China.

A Gram-negative, aerobic, non-motile, rod-shaped bacterial strain, designated 25-1(T), was isolated from the air inside giant panda enclosures at the Chengdu Research Base of Giant Panda Breeding, China. Strain 25-1(T) grew optimally at pH 7.0-8.0, at 28-30 °C and in the presence of NaCl concentrations from 0.0% to 0.5 %. 16S rRNA gene sequence analysis indicated that strain 25-1(T) belongs to the genus Chryseobacterium within the family Flavobacteriaceae and is related most closely to C. carnis G81(T) (96.4% similarity), C. lathyri RBA2-6(T) (95.8% similarity), and C. zeae JM1085(T) (95.8% similarity). Its genomic DNA G+C molar composition was 36.2%. The major cellular fatty acids were iso-C15:0 (44.0%), iso-C17:0 3OH (19.8%) and C16:1 ω7c/16:1 ω6c (12.7%). The only isoprenoid quinone was menaquinone 6 (MK-6). The major polar lipids were phosphatidylethanolamine, two unidentified amino lipids and two unidentified lipids. The DNA-DNA relatedness between strain 25-1(T) and C. lathyri RBA2-6(T) was 38%. Phenotypic, genotypic, and phylogenetic characteristics indicated that strain 25-1(T) is a novel member of the genus Chryseobacterium, for which the name C. chengduensis sp. nov. is proposed. The type strain is 25-1(T) (CCTCC AB2015133(T)=DSM 100396(T)).

Effects of different starch sources on Bacillus spp. in intestinal tract and expression of intestinal development related genes of weanling piglets.

The study was conducted to evaluate the effects of different starch sources on Bacillus spp. in intestinal tract and expression of intestinal development related genes of weanling piglets. Twenty-eight PIC male piglets were divided into four homogeneous groups according to initial body weight (similar birth and parity, weaned at 21 ± 1.5 days). Diets for the four treatments consisted of corn starch, wheat starch, tapioca starch and pea starch with the determined ratio for amylose to amylopectin of 0.21, 0.24, 0.12 and 0.52 respectively. Real-time quantitative polymerase chain reaction was applied to: (1) detect genomic DNA of Bacillus and to quantify the number of Bacillus in the intestinal tract chyme of piglets with the primers and probe which designed based on the 16S rRNA sequences of maximum species of Bacillus on GenBank; (2) measure the mRNA level of glucagon-like peptide 2 (GLP-2), insulin-like growth factors 1 (IGF-1) and epidermal growth factor (EGF) in duodenum, jejunum and ileum. Results showed that the number of Baciilus and the percentage based on all bacteria in the whole intestinal content of piglets fed pea starch was highest in all groups (P < 0.05). There was no significant differance on copy numbers for all bacteria and Bacillus in the whole intestinal tract of piglets between the corn starch group and wheat starch group (P > 0.05). In addition, the expression level of GLP-2, IGF-1 mRNA in jejunum and ileum of pea starch treatment (the high amylose/amylopectin ratio) were increased while the tapioca starch decreased their mRNA level significantly compared to other three treatments (P < 0.05). There was no significant difference for the mRNA level of EGF in each group. The present study revealed that high amylose/amylopectin ratio of starches significantly enhanced the numbers of Bacillus in all segments of intestine and the mRNA level of intestinal development related genes.

Design, expression and characterization of recombinant hybrid peptide Attacin-Thanatin in Escherichia coli.

Antimicrobial peptides will be attractive and potential candidates as peptide drugs because of their efficient action against microbes and low toxicity to mammal cells. To improve their antibacterial activity, some modifications needs to be made. In this research, the hybrid peptide gene Attacin-Thanatin with 642 bp in length with preferred codons of E. coli was generated using the technology of Gene splicing by overlap extension. The gene was inserted in-frame into E. coli expression plasmid pET-32a (+) and induced to express in E. coli Rosetta. The recombinant protein was partial purified and its biological activity was determined. Analysis of the E. coli Rosetta induced with IPTG revealed that the molecular weight of fusion protein was approximately 41.8 kDa, which perfectly matched the mass calculated from the amino acid sequence. Biological activity detection showed that this peptide effectively inhibited the growth of the test bacteria including E. coli DH5α, E. coli BL21 (DE3), Salmonella choleraesuis and Staphylococcus aureus. Among these bacteria, the Gram-negative E. coli was the most sensitive. Furthermore, there was minor hemolysis activity for porcine red blood cells. So, the results indicated that the hybrid peptide Attacin-Thanatin could be served as a promising candidate for the chemical antibiotics.

Molecular cloning, expression in Escherichia coli of Attacin A gene from Drosophila and detection of biological activity.

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS-PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS-PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G- bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.