Complete genome sequence and proteomic analysis of a thermophilic bacteriophage BV1

Viruses of thermophiles are of great interest due to their roles in gene transfer, global geochemical cycle and evolution of life on earth. However, the thermophilic bacteriophages have not been studied extensively. In this investigation, a typical bacteriophage BV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k512, which was isolated from an inshore hot spring in Xiamen of China. The BV1 contained a double-stranded linear DNA of 35 055 bp, which encodes 54 open reading frames (ORFs). Interestingly, eight of the 54 BV1 ORFs shared sequence similarities to genes from human disease-relevant bacteria. Seven proteins of the purified BV1 virions were identified by proteomic analysis. Determination of BV1 functional genomics would facilitate the better understanding of the mechanism for virus-thermophile interaction.     

噬菌体在水产养殖中治疗细菌感染的研究进展和挑战

高密度、集约化水产养殖业的发展导致疾病不断发生并呈现日趋严重之势,同时,由于细菌对传统抗生素产生耐药性而使抗生素的使用受到越来越多的限制,世界各国科学家都在致力于研究替代抗生素的新疗法。噬菌体因为可以特异性地杀死某些细菌,对动、植物以及环境都没有副作用,因此,在水产养殖病害防治中呈现良好的应用前景。本文从噬菌体的特异性、安全性、赋形方法以及对环境的影响等方面对国内外水产养殖中的噬菌体治疗研究现状和存在的主要问题进行了简要论述,为了解学科进展,促进噬菌体在治疗中的成功使用提供了理论依据。     

Properties of Klebsiella phage P13 and associated exopolysaccharide depolymerase

The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70℃ for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60℃ and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.     

Use of N stable isotope and microbial analyses to define wastewater influence in Mobile Bay, AL

We assessed short-term ecological and potential human health effects of wastewater treatment plant (WTP) effluent by measuring δ¹⁵N‰ and microbial concentrations in oysters and suspended particulate matter (SPM). We also tested male-specific bacteriophage (MSB) as an alternative to fecal coliforms, to assess potential influence of wastewater contamination on shellfish. WTP effluent did not affect oyster growth or survival, but SPM and oysters acquired wastewater-specific δ¹⁵N‰. δ¹⁵N values were depleted near the WTP, typical of low-level processed wastewater. Fecal coliform and MSB concentrations were higher in samples taken closest to the WTP, and MSB values were significantly correlated with δ¹⁵N‰ in oyster tissues. Overall, oysters demonstrated relatively rapid integration and accumulation of wastewater-specific δ¹⁵N‰ and indicator microorganisms compared to water samples. These data suggest oysters were superior sentinels compared to water, and MSB was a more reliable indicator of wastewater influence on shellfish than fecal coliforms.     

深海噬菌体BVE2内溶素的重组表达及其活性研究

本研究从一株来源于西南印度洋沉积物的噬菌体BVE2基因组中得到了一条全长702 bp,编码噬菌体内溶素的基因Lysin132,编码的蛋白共含有234个氨基酸残基,预计分子量为25.74 kDa;氨基酸序列同源比对结果表明BVE2 Lysin132具有多个酰胺酶活性位点。进化树分析结果表明BVE2 Lysin132是一种N-乙酰胞壁酰-L-丙氨酸酰胺酶。将Lysin132克隆到pEASY-Blunt E2 Expression Vector中,构建了E.coli-pEASY-Lysin132表达菌株。成功用IPTG诱导表达,得到了大量带有6×His标签的重组BVE2 Lysin132。经镍柱纯化后进行SDS-PAGE电泳,得到单一目的蛋白条带。酶学性质分析结果表明,重组BVE2 Lysin132的最适反应温度为30℃,在中高温下热稳定性良好,在10～50℃下孵育90 min,仍能保持70%以上的酶活力;最适pH为7.0,在pH 6.0～8.0时均能保持80%以上的酶活力,表明该酶有较宽的pH作用范围;Na⁺、Mg²⁺可以使酶活力显著提高20%以...     

Modification of the polyethylene glycol 6000 precipitation method for recovering human and indicator viruses from oysters and mussels

Human viruses are a common contaminant of shellfish affected by human sewage wastes. They are difficult to detect as they are not easily separated from shellfish tissue. This paper describes a modification of the polyethylene glycol (PEG) precipitation technique for recovery of enteroviruses and F‐specific bacteriophages from the Pacific oyster (Crassostrea gigas) and the green‐lipped mussel (Perna canaliculus). Modifications adopted were the use of only the digestive gland tissue for virus extraction, resuspension of the primary PEG pellet in 4 volumes of eluent, and the introduction of a secondary PEG precipitation to reconcentrate the virus containing extract. The recovery rate of the virus extraction process was not affected by introduction of the secondary concentration step (overall recovery remained at 60–70% of the virus input). The advantages of reduction of tissue residue in the extract, smaller final volume, and the ability to process 2–3 times the number of individual shellfish for the same effort, improve the practicality of the method.     

Five new species of Halacaridae (Acari) from New Zealand

In the Cape Rodney to Okari Point Marine Reserve, Goat Island Bay, Auckland, New Zealand, several halacarids were collected on an intertidal flat. The mites were found associated with encrusting bryozoans, sponges, and lithothamnia. Five mites—Rhombognathus lacunosus n.sp., Rhombognathus fractus n.sp., Halacarus nitidus n.sp., Agauopsis similis n.sp., and Agaue insignata n.sp.—are described, illustrated, and compared with related species.