Purification and characterization of carbon monoxide dehydrogenase from the aerobic hyperthermophilic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis>

The aerobic hyperthermophilic archaeon Aeropyrum pernix expresses carbon monoxide (CO) oxidation activity under heterotrophic growth conditions. Using activity stain gel analysis, CO oxidation activity was detected in a protein with a molecular mass of 210 kDa. The 210 kDa CODH protein was purified to homogeneity from A. pernix. Aeropyrum Mo-CODH catalyzed the oxidation of CO with a specific activity of 2.1 μmol CO min⁻¹ mg⁻¹ at 95°C, pH 8.0 using methyl viologen as the electron acceptor. The CODH protein showed high oxygen and thermo stability. The protein contains three subunits: L (86.6 kDa), M (34.5 kDa), and S (12.6 kDa), which form the LM₂S complex. The molecular mass of the complex was calculated by gel filtration and found to be 163.7 kDa. N-terminal amino acid sequencing and peptide mass fingerprinting analysis of the subunits indicated that they corresponded to NP_148462.1, NP_148464.2, and NP_148465.1, and their genes annotated the molybdo iron-sulfur flavoprotein carbon monoxide dehydrogenase S, L, and M subunits, respectively. Phylogenetic analysis revealed that CODH belongs to a novel clade of diverse CODHs.     

一种免疫诱导型鲤启动子的克隆与功能分析

为发掘适用于基因工程抗病育种的鱼类启动子,通过实时荧光定量PCR实验对鲤Rab GTP酶(Ras-associated binding-GTPases 1a3,Rab1a3)基因的表达模式进行了分析,证实该基因在鳃、头肾等与机体免疫防御功能密切相关的组织内转录水平较高,且免疫激活后转录显著增强,符合基因工程抗病育种所需的外源免疫基因转录模式。从鲤细菌人工染色体文库中,使用Rab1a3基因特异引物筛选获得包含该基因区域的文库克隆,测序获得该基因完整序列,以及上下游调控序列。通过生物信息学手段,预测到长度为1014 bp的鲤Rab1a3基因启动子序列,该启动子不具有典型的TATA盒或CpG岛特征,存在多个免疫相关转录因子结合位点。在草鱼肾组织细胞系内验证该启动子活性,结果显示,绿色荧光蛋白基因和萤火虫荧光素酶基因都能够在该启动子驱动下表达,证实该片段具有启动子活性,且启动子活性在受到免疫诱导后增强,双荧光素酶报告基因检测结果显示,该启动子活性在免疫刺激后增强至免疫刺激前的8.67倍。研究表明,鲤Rab1a3基因启动子有望被开发成为免疫诱导型的基因工程元件,驱动外源免疫基因在鱼体内适时表达,抵御外界病原感染,同时避免非必要条件下的过度表达形成生长负担。     

Non-specific immune response of Nile tilapia,Oreochromis nilotica,to the extracellular products of Mycobacterium spp. and to various adjuvants

Nile tilapia were immunized by injecting extracellular products (ECP) of Mycobacterium spp. (strain TB40, TB267 or the type strain Mycobacterium marinum) into their swim bladders. A variety of adjuvants – Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and Titremax – were similarly injected into additional groups of tilapia. Phosphate-buffered saline (PBS) was used as a control. The number of nitroblue tetrazolium (NBT)-positive cells observed in the swim bladder of the immunized fish had significantly increased by the fourth day post-immunization. By day 8, the number of NBT-positive cells in fish immunized with ECP from mycobacteria strains TB40 or TB267 were fewer than in fish immunized with ECP from M. marinum or fish injected with FCA or FIA. The level of lysozyme activity detected in the serum of fish 4 days after being immunized with ECP from various Mycobacterium spp. was also significantly higher than that found in the serum of the control fish. Head kidney macrophages showed an enhanced reduction of NBT when cultured in vitro with 1 μg ml^–1 of ECP. Concentrations greater than this (10 or 100 μg ml^–1) were found to suppress the reduction of NBT by the macrophages.     

Genetic variation in mitochondrial genes and intergenic spacer region in harmful algae <Emphasis Type="Italic">Chattonella</Emphasis> species

In this study, nuclear ribosomal RNA gene internal transcribed spacer regions and the cox2-cox1 fragment of the mitochondrial (mt) genome were sequenced in 24 strains of Chattonella spp. Variability in both regions showed that the mt genome sequences of Chattonella spp. have a higher evolutionary rate than the nuclear rRNA gene sequences. A maximum likelihood tree based on the mt sequence grouped the Japanese Chattonella strains into two groups (Groups A and B), although no correlation was observed amongst the phylogenetic groups, their morphologies, or the isolated areas. Groups A and B were clearly identified by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using Fokl, without the need for a sequencing experiment. The PCR-RFLP assay revealed that Chattonella cells obtained from sea water in Oita, Japan, in 2004 and 2005 belonged to Group B. This is the first report showing the genetic variation in Chattonella spp. using a PCR-RFLP identification protocol.     

Molecular divergence and application of the ITS-5.8S rDNA and RUBISCO spacer in <Emphasis Type="Italic">Porphyra haitanensis</Emphasis> Chang et Zheng (Bangiales,Rhodophyta)

To select a reliable and sensitive method for discriminating strains of Porphyra haitanensis, the nucleotide sequence of the internal transcribed spacer 1 to internal transcribed spacer 2 regions (ITS-5.8S) of nuclear ribosomal DNA and the intergenic spacer region of RUBISCO were compared in five wild and five cultivated Porphyra haitanensis strains. Based on molecular analyses, sequences of ITS-5.8S (about 1,210 bp) could be divided into three regions: ITS1, 5.8S, and ITS2. The ITS1 and ITS2 sequences of each strain differed, even between individuals collected from the same site. In contrast, 5.8S rDNA and RUBISCO spacer sequences were identical among the ten P. haitanensis strains, although differences were found among different Porphyra species. Phylogenetic analysis also supported these conclusions. These sequence features of highly conserved regions and diversified regions that occurred repeatedly in ITS-5.8S could be useful in discriminating germplasm of P. haitanensis strains or Porphyra species. In contrast, the RUBISCO spacer is only suitable for identifying Porphyra species. New coupled primers were designed to amplify only the 5.8S rDNA and ITS2 region of Porphyra. The sequences of these amplified fragments can be readily used to identify germplasm or to perform phylogenetic analysis of Porphyra spp.     

凡纳滨对虾肠道内产消化酶益生菌的分离与筛选

为获得具有消化酶活性且安全的益生菌,从凡纳滨对虾肠道中初步分离得到576株细菌,对菌株进行产蛋白酶、淀粉酶和脂肪酶能力的定性及定量测试,筛选出产酶种类多且产酶能力强的菌株11株。对筛选出的11株菌进行了幼虾浸浴实验、药敏性实验和溶血性实验,以评价其生物安全性。将11株菌的菌悬液添加到凡纳滨对虾幼虾的养殖水体中进行浸浴实验,浸浴结束后用鳗弧菌进行刺激,测定不同浸浴组幼虾相关免疫基因的相对表达量,以确定其对幼虾的保护效果。综合消化酶活性、菌株对幼虾的保护效果及生物安全性,筛选得到4株效果较好的菌株。菌株的16S r DNA分子鉴定结果表明,细菌1号、2号和4号分别与芽孢杆菌(Bacillus sp.PCSAS2-38,GQ284495.1)、蜡样芽孢杆菌(B.cereus strain N419,JN400121.1)及苏云金芽孢杆菌(B.thuringiensis strain EA26.1,KC758847.1)的相似性均为100%,9号菌株与荚膜红细菌(Rhodobacter capsulatus strain PSB-03,FJ866782.1)相似性达到99%,为后续益生菌制剂的开发奠定了前期基础。     

Bacteria associated with early life stages of the great scallop, <Emphasis Type="BoldItalic">Pecten maximus</Emphasis>: impact on larval survival

A bacteriological study was carried out at a scallop (Pecten maximus) hatchery near Bergen, western Norway following a severe increase in mortality rates during the larval stages of the scallops. No larvae survived to settling, except for those in groups treated prophylactically with chloramphenicol. In order to identify pathogenic strains of bacteria, we performed a challenge test on 10- to 16-day-old larvae using isolated bacterial strains from the hatchery. Infection with six of these strains produced mortalities that were not statistically different from that resulting from infection with the known pathogen Vibrio pectenicida. However, about 5% of the strains tested in the challenge experiment produced higher motility rates than found in the unchallenged control group, indicating a possible probiotic effect. On the basis of 16S rDNA analysis on these strains, the phylogenetic tree indicated two groups of apparent pathogens: (1) one strain, LT13, grouped together with Alteromonas/Pseudoalteromonas; (2) a cluster of strains grouped together with Vibrio splendidus (LT06, LT21, LT73, PMV18 and PMV19). Strain LT13 was isolated from cultures of the microalga Chaetoceros calcitrans used for feed, while the other strains were isolated from larval cultures. Transmission electron microscopy showed intracellular bacteria that resembled bacteria in the groups Chlamydiaceae and Rickettsiaceae.     

Isolation and evaluation of putative probiotic strains from different teleost to prevent Pseudomonas aeruginosa infection in Cyprinus carpio

Probiotics renowned as valuable microbes serve as a potential alternative to control diseases in aquaculture and are considered as an efficient and environment‐friendly approach to reduce the use of antibiotics. The present study aims at the isolation of putative probiotic bacteria from the intestinal tract of different fish species from the Doaba region of Punjab, India. In this study, isolated bacterial strains were characterized based on their morphological, biochemical and molecular characterization by 16S rRNA gene sequencing, followed by in vitro evaluation of different selection parameters described in FAO/WHO guidelines. A total of 169 different bacterial strains were isolated from the gastrointestinal tract of 52 different fish species. After in vitro evaluation, out of 169 bacterial strains only five bacteria (S3, S7, BDK2', BDK7 and BDK9) identified as Enterococcus and Bacillus species showed antagonistic activity against the fish pathogen Pseudomonas aeruginosa (MTCC 4 673). These isolates were screened based on their response towards bile tolerance, pH tolerance, adhesion and drug susceptibility to different antibiotic discs. And, the in vivo results indicated improved growth and survival against the infection (P. aeruginosa) after oral administration of the probiotics. The observations of in vitro and in vivo evaluation indicate that these isolated probiotic strains serve as effective probiotics and can be used as a novel and safe treatment to cure current issues in aquaculture.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

Extracellular products from Mycobacterium spp. in fish

Mycobacterium spp. isolated from food and ornamental fish in Thailand (TB1, TB40, TB267 and TB268), and the type strains Mycobacterium marinum (NCIMB 1298), M. fortuitum (NCIMB 1294) and M. chelonae (NCIMB 1474) were cultured in Long's medium, Eagle's minimum essential medium, Sauton's medium and modified Sauton's medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB 40, TB267, TB268 and M. marinum NCIMB 1298 in particular, whereas growth and production of ECP for all strains was limited in Long's medium. SDS-PAGE protein profiles of ECPs from 14-day culture supernatants showed major bands at 65 and <14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M. marinum averaged approximately four- to 10-fold higher than from strains cultured for 14 days at 28°C. Enzyme testing for the type strains indicated only mucinase activity for M. marinum, while lipase and RNase activities were detected for M. chelonae and M. fortuitum . Protease and DNase activities could not be detected for any of the Mycobacterium spp. tested. The medium lethal dose (LD₅₀) of ECP to rainbow trout and Nile tilapia was greater than 400 μg protein fish^–1.     

Probiotic activity of bacteria associated with egg capsules of Concholepas concholepas (common name ‘Loco’)

Egg capsules of Concholepas concholepas were analysed microbiologically, showing the presence of bacteria inside the capsules. The bacteria were isolated and identified, and their probiotic potential on early larvae of C. concholepas was evaluated. From 32 capsules that were examined, 45 bacterial strains were isolated, of which 67% belonged to the genus Bacillus. Survival bioassays were carried out with all the isolated bacterial strains on C. concholepas larvae. It was found that 71% of the bacteria had positive effects on larval survival. This research determined that bacteria of the genera Bacillus and Vibrio present in the capsules have probiotic potential. The bacteria that led to the greatest larval survival were Bacillus pumilus, which may be used in the cultivation of C. concholepas, because one of the limiting factors for the cultivation of this species is larval survival; thus, we propose that the probiotic strains identified in this study will be used during the cultivation of C. concholepas to enhance the chances of larval survival.     

Microsatellite marker isolation and cultured strain identification in <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.