嗜冷黄杆菌酰基-Acp去饱和酶（AAD）的序列分析和结构建模

多不饱和脂肪酸是生物膜的重要组成成分,在维持细胞膜的流动性与低温适应性中起到重要作用。以低温微生物嗜冷黄杆菌为对象,主要采用多重序列比对、同源建模、模型评价等生物信息学技术与方法,分析与研究了多不饱和脂肪酸代谢重要相关蛋白酰基-Acp去饱和酶的序列组成与基序特征、系统进化与重要活性位点中心,模建并高评分地获得了其三维空间结构。系统发育分析表明,嗜冷黄杆菌等低温耐受菌与植物类物种的酰基-Acp去饱和酶的分子进化关系较近,而与其他细菌的分子进化关系较远。多序列基序分析显示,多数物种含有HxxxxH、HxxHH和HxxHH等3个组氨酸保守区,为二Fe离子结合区。Swiss-model对嗜冷黄杆菌的酰基-Acp去饱和酶进行的同源建模及其模型分析发现,其也具备与Fe离子相互作用的活性区,与Fe离子直接作用的活性位点有His114、Glu111、Glu76、His200、Glu19和Glu164,嗜冷黄杆菌的酰基-Acp去饱和酶有异于其它细菌的特异性。这从某种程度上揭示,相对于常温菌而言,低温耐受菌可能存在着低温相关蛋白酶的序列乃至功效上的差异。对于深入了解细菌耐低温机制,低温相关蛋白差异性分析及改造与应用均有重要的意义与价值。     

Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum

To go further into the characterization of the proteolysis exocellular system of the salmonid pathogen Flavobacterium psychrophilum, the purification and characterization of a novel protease designated Fpp2 (F. psychrophilum protease 2) was undertaken. A protease (Fpp2) hydrolyzing azocasein was purified. The Fpp2 can be defined as a metalloprotease, it had an estimated molecular mass of 62 kDa with calcium playing an important role in the thermostability of the enzyme. Proteolytic activity was optimal at pH 6.0-7.0 and 24 degrees C and activation energy for the hydrolysis of azocasein was determined to be 5.4 kcal mol(-1), being inactive at temperatures above 42 degrees C. All these results are characteristic of 'cold adapted enzymes'. Fpp2 proved to be a broad range hydrolytic enzyme because in optimal conditions it was able to hydrolyze matrix and muscular proteins. It can be concluded that the Fpp1, a previously characterized 55 kDa metalloprotease, and the Fpp2 protease were produced under different physiological conditions and were immunologically as well as biochemically different.     

Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum

AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues. METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl. psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions. Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass. The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl. psychrophilum tested here. CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e. S-layer), one of the most common surface structures on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface.     

The Flavobacterium psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in rainbow trout

AIMS: The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in a possible vaccine. METHODS AND RESULTS: The expression product of ompA is a 465-amino-acid protein precursor that contains a 21-amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy-terminal region contains the 'OmpA/MotB' domain/signature and five putative 'Thrombospondin type 3 repeats' domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti-OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund's adjuvant developed a high antibody titter. CONCLUSIONS: Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine.     

Purification and characterization of a membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum

AIMS: The cell envelope of the fish pathogen Flavobacterium psychrophilum contains more than 50 polypeptides resolved by sodium dodecyl sulphate-polyacrylaminde gel electrophoresis analysis including a major component named P60. Here, we have developed a simple and efficient procedure for the purification of P60 and therefore permitting its biochemical characterization. METHODS AND RESULTS: Membrane proteins were selectively extracted from isolated cell envelopes with the mild non-ionic detergent Triton X-100. About 10 polypeptides were identified from the detergent fraction, including P60. The P60-enriched fraction was thereafter subjected to an anion exchange chromatographic step in the presence of Triton X-100. The molecule was purified at the milligram level (yield, about 75%; purification factor, 6.2). Analyses performed by charge shift electrophoresis, Triton X-114 phase separation and by detection of sugar-modified components showed that P60 is a true amphiphilic membrane-associated glycoprotein. CONCLUSIONS: The method described in this paper provides pure and non-denaturated P60 and should prove to be easily scaled-up. As sugar-modified protein, P60 should be included in the growing list of glycosylated prokaryotic proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: It offers the possibility of obtaining P60 in amounts allowing the testing of the potential of P60 as a candidate for anti-flavobacteria subunit vaccines, as P60 is one of the major antigens.     

The use of warmed water treatment to induce protective immunity against the bacterial cold-water disease pathogen Flavobacterium psychrophilum in ayu (Plecoglossus altivelis)

We investigated the induction of protective immunity against bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum by warmed water treatment in ayu (Plecoglossus altivelis). Fish were immersed in a live bacterial suspension (10⁷ CFU mL⁻¹) for 30 min and placed in 700 L concrete tanks. The 28 °C warmed water treatment lasted 3 days and began 1, 6, and 24 h after immersion in the live bacterial suspension. A naïve control fish group was immersed in a sterilized modified Cytophaga (MCY) broth instead of the bacterial suspension. Fourteen days after the immersion, agglutination antibody titers against F. psychrophilum were measured by using micro-titer methods. Fish were then exposed to a bacterial bath to infect them with live F. psychrophilum, and cumulative mortality was monitored. Fish treated with warmed water at 1, 6, and 24 h after immersion in the live bacterial suspension had cumulative mortalities of 36%, 30%, and 18%, respectively, all of which were significantly lower than the cumulative mortality of the naïve control fish (90%). Treated fish also showed high antibody titers against F. psychrophilum in agglutination tests. These results demonstrate that warmed water treatment could not only cure BCWD but also immunize the fish against the causative agent F. psychrophilum.     

Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum     

Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium

A cryptic plasmid found at high copy number was isolated from Flavobacterium sp. KP1, a psychrophilic Gram-negative bacterium, cloned, and sequenced. The sequence will appear in the DDBJ/EMBL/GenBank databases under the accession number AB007196. The pFL1 plasmid is 2311 nucleotides in length with 32.7% GC content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. The plasmid contains two open reading frames of significant length, ORFI and ORFII. ORFI encodes a protein similar to the replication proteins found in Gram-negative bacterial plasmids, Bacteroides fragilis plasmid pBI143 and Zymomonas mobilis plasmid pZM2. The putative translation product of ORFII shows homologies with plasmid recombination proteins found mainly in Gram-positive bacterial plasmids such as Staphylococcus aureus plasmid pT181.     

Isolation of a gene encoding cysteine synthase from Flavobacterium K3–15

Abstract The cysteine synthase gene ( cysK ) from Flavobacterium K_3–15 was cloned and sequenced. The gene exhibits 30–50% identity to known cysteine synthases on both the DNA and the amino acid levels. The pyridoxal phosphate binding site of the enzyme is part of a conserved motif comprising seven amino acids (SIKDRIA). The lys31 residue of the flavobacterial enzyme is conserved in all known cysteine synthases. The cysK gene from Flavobacterium K_3–15 was heterologously expressed and the gene product identified by immunoblotting and determination of the enzyme activity.     

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.     

Separation of viable from nonviable cells using a discontinuous density gradient

Summary When a suspension containing a mixture of viable and nonviable cells is layered over a dense ficoll-metrizoate solution and centrifuged, most of the viable cells are retained at the interface above the dense solution; whereas most of the nonviable cells are distributed in other fractions. The cells revovered for the interface are capable of subsequent growth in culture.     

Microbiological removal of pentachlorophenol from soil using a Flavobacterium

Experiments reported here show that it is possible to remove pentachlorophenol (PCP) from a variety of contaminated soils, including actual waste-dump soils, by inoculating such soils with cells of a PCP-degrading Flavobacterium. However, soil conditions such as temperature, water content, PCP concentration and density of bacterial cells must be maintained within optimum ranges. Manipulation of the natural microflora so that it degrades PCP may be the preferred decontamination method in many instances. Very highly contaminated soils are so toxic that methods such as soil leaching followed by decontamination of the leachate may be required, as direct inoculation or self-purification are ineffective.     

Recovery of plant DNA using a reciprocating saw and silica‐based columns

The time needed for hand grinding and the cost of commercially available extraction kits remain to be the major limitations in plant DNA extraction for many researchers. We present inexpensive techniques for (i) simultaneously machine grinding large numbers of plant samples for DNA extraction using a commercially available reciprocating saw; and (ii) DNA recovery using silica column‐based extractions similar to that used in some commercially available kits. Used together, these allow for the rapid recovery of plant DNA at relatively low cost. Furthermore, these methods appear to be widely applicable within plants with good yields recovered in test extractions across major plant groups (ferns, gymnosperms, monocots and eudicots).     

Resuscitation of viable but non-culturable Vibrio parahaemolyticus in a minimum salt medium

Vibrio parahaemolyticus is food-borne pathogen prevalent in Asian countries. This work analyzes factors that influence the resuscitation of the viable but nonculturable (VBNC) state in this bacterium. The MMS-0.5% NaCl medium alone limited cell multiplication, and in this medium, resuscitation was successful when the temperature was upshifted to 25 degrees C but not 37 degrees C. Chloramphenicol inhibition experiments revealed that protein synthesis in the first 24 h of temperature upshift was critical in determining the success of the three-day resuscitation period. The VBNC state induction period and the age of the VBNC cells for successful resuscitation were strain-dependent. Results of this work facilitate further physiological and pathological study of the VBNC state in this pathogen.     

Recovery of insect cells using hollow fiber microfiltration

An efficient method was developed for media separation and cell collection for eukaryotic cells growing in suspension. The method is based on tangential flow microfiltration using an open channel arrangement in a hollow fiber configuration. Best results (highest processing flux rate) for polysulfone hollow fibers were obtained using fibers with internal diameter of 0.75 mm, 0.45 mum pore size, and a cell suspension flow at a shear rate of 14000 s(-1) (0.032 L/min per fiber). A flux rate of 500 L/m(2) h can be obtained by maintaining the surface area/cell ratio at 0.05 m(2)/10 L of cells at a concentration of 2.5 x 10(6) cells/mL. Forty liters of infected insect cells can be concentrated 10 times in 20 min without affecting cell viability. (c) 1995 John Wiley & Sons, Inc.     

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.