Molecular cloning and nucleotide sequence analysis of the maltose-inducible porin gene of Aeromonas salmonicida

Abstract The gene for the Aeromonas salmonicida maltose-inducible porin (maltoporin) was cloned into phagemid pTZ18R in two restriction fragments, 0.6-kb Pst I/ Kpn I and 1.7-kb Sph I, of genomic DNA and their nucleotide sequences were determined. Open reading frames of 1329 and 1335 bp translated into sequences of 443 and 445 amino acids, with a 23 or 25 amino acid signal sequence and a 420 amino acid mature protein of molecular mass 46424 Da. Putative ribosome binding sites, AGGA and GGGAA, occurred 9 bp upstream of two possible ATG initiation codons. The A. salmonicida gene product showed a high degree of similarity with Escherichia coli LamB, and codon usage was very similar to that of another A. salmonicida outer membrane protein but markedly different from those of extracellular proteins.     

In vivo production of A-protein, lipopolysaccharide, iron-regulated outer membrane proteins and 70-kDa serine protease by Aeromonas salmonicida subsp. salmonicida

Using specific immunostaining of Western blots, the in vivo expression of several putative virulence factors of Aeromonas salmonicida subsp. salmonicida was demonstrated in infected muscle tissue of Atlantic salmon and rainbow trout. Three virulent isolates of A. salmonicida were used. One isolate was chosen because in vitro it was apparently a non-producer of the 70-kDa serine protease. Infected furuncle tissue was centrifuged and samples of the pellet and supernatant probed for evidence that the components of interest were bacterial cell-associated or secreted. The A-protein was detected in pelleted furuncle material but not in the supernatant. Lipopolysaccharide, both high and low molecular mass, was present in the pellet but only high molecular mass lipopolysaccharide was detected in the furuncle supernatant. Iron-regulated outer membrane proteins were detected in the furuncle pellet. The 70-kDa serine protease was detected in the furuncle supernatant of both protease-producing strains. However, whilst the protease-deficient isolate was demonstrated to produce low levels of the 70-kDa protease when grown in vitro under iron restricted conditions, none could be detected in vivo.     

Pulsed-field gel electrophoriesis analyis of Aeromonas salmonicida ssp. salmonicida

A total of 133 strains of Aeromonas salmonicida ssp. salmonicida, isolated from a wide variety of sources, were characterized by pulsed-field gel electrophoresis patterns. Sixteen profiles were demonstrated, with one profile being predominant in samples from all the countries and species of fish. Our results suggest a clonal distribution of this subspecies, with a predominant clone being responsible for most of the outbreaks worldwide.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

The quantitative relationship of lethality between extracellular protease and extracellular haemolysin of Aeromonas salmonicida in Atlantic salmon (Salmo salar L.)

An additive relationship of lethality between purified protease and haemolysin of the extracellular products (ECP) of Aeromonas salmonicida was demonstrated by i.p. injection in Atlantic salmon (Salmo salar L.). The lethal toxicity of the combinations of protease and haemolysin follow a linear regression line y = -54.54x + 2400. The LD50 of protease and haemolysin when injected separately was 2400 ng/g fish and 44 ng protein/g fish, respectively.     

异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

Evidence against dormacy in the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida

Results indicate that A. salmonicida does not enter an unculturable dormant state. The resuscitation of dormant cells by nutrient broth described by previous workers appears to be due to the presence of small numbers of viable, culturable cells too few to be detected by the sampling protocol employed.     

Survival of the fish pathogen Aeromonas salmonicida in seawater

Survival of Aeromonas salmonicida in natural (non-sterile) seawater, as determined from colony counts on marine agar, was found to be influenced by the presence of potentially inhibitory organisms, i.e., Acinetobacter, Aeromonas hydrophila, Chromobacterium, Escherichia coli, Flavobacterium and Pseudomonas, and their metabolites. Yet, samples, thought to be devoid of culturable A. salmonicida, were found to contain cells, which were filterable through 0.22 and 0.45 microns Millipore Millex porosity filters, and were recoverable on a specialised medium for L-forms, i.e. L-F medium.     

Partial purification and properties of a haemolytic activity (T-lysin) from Aeromonas salmonicida

Abstract An extracellular haemolytic activity, produced by the fish pathogen Aeromonas salmonicida , against trout erythrocytes (T-lysin) was partially purified by ammonium sulphate precipitation and gel chromatography. Lysis of trout erythrocytes was found to be due to the combined activity of a caseinase and another factor, apparently membrane-associated, which when separated caused incomplete lysis. Incomplete lysis was also observed when caseinase production was suppressed by the incorporation of ammonium sulphate in the growth medium, or in caseinase-negative mutants. Inhibition of caseinase activity by phenyl methyl sulphonyl fluoride also resulted in the loss of full lytic potential from culture supernates containing T-lysin.     

Characterisation of an extracellular serine protease gene (nasp gene) from Dermatophilus congolensis

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.     

Identification of serine protease,serine protease homolog and prophenoloxidase genes in Spodoptera frugiperda (Lepidoptera: Noctuidae)

In insects, proteolytic cascades medicated by serine proteases (SPs), serine protease homologs (SPHs) and prophenoloxidases (PPOs) control several physiological processes, notably the innate immunity. However, no attempts have been made to identify and characterize these genes in Spodoptera frugiperda, one of the most destructive agricultural pests. In this study, 83 SPs, 26 SPHs and four PPOs were respectively identified in S. frugiperda genome based on homology blast against those of other insects. We then analyzed the domain organization of these proteins and assigned them into different groups by phylogenetic reconstruction. Furthermore, the mRNA levels of clip-domain SPs/SPHs (cSPs/cSPHs) and PPOs were quantified in response to a mixed infection of Micrococcus luteus and Escherichia coli, and obvious accumulations were recorded in immune tissues, including hemocytes and fat body. In the latter study, we profiled the expression patterns of highly expressed cSPs and PPOs in different developmental stages, including egg, larva, pupa, female and male adults. It was shown that most cSPs were abundantly expressed in adults, while PPOs were detected at high levels in both egg and larval stages. These current findings substantially add to our understanding of the roles of S. frugiperda SPs, SPHs and PPOs in immune regulation and further lay a solid foundation for uncovering the interaction mechanisms between insects and pathogens.     

The carboxy‐terminal tail of Aeromonas sobria serine protease is associated with the chaperone

ASP is the only bacterial protease in the kexin group of the subtilisin family. Previous studies have revealed that the ORF2 protein encoded at the 3′ end of the asp operon is required for ASP to change from a nascent form into an active form in the periplasm. However, the mechanism by which ORF2 makes contact and interacts with ASP in the maturation process remains unknown. The present study examined the effect of mutations in the carboxy‐terminal region of ASP on the ASP maturation process. Both deletion‐mutation and amino acid‐substitution studies have demonstrated that the histidine residue at position 595 (His‐595), the sixth residue from the carboxyl terminus of ASP, is highly involved in the generation of active ASP molecules. An analysis by pull‐down assay revealed that mutation at His‐595 reduces the efficacy of nascent ASP to transition into active ASP by reducing the ability of ASP to make contact and interact with ORF2. Thus, it appears likely that nascent ASP in the periplasm interacts with ORF2 via the carboxy‐terminal region, and His‐595 of ASP appears to be an indispensable residue in this interaction.     

Starvation survival of the fish pathogen Aeromonas salmonicida in seawater

Abstract Three strains of the fish pathogenic bacterium Aeromonas salmonicida were examined with respect to their ability to survive in seawater. Five to seven days after plate counts decreased below the detection limit of 10 cells/ml, the population of respiring A. salmonicida cells comprised less than 4% of the initial total bacterial population. At this stage, samples were transferred either to sterile nutrient or to control flasks containing sterile nutrient-free medium. The addition of nutrients caused reappearance of growing bacteria, detected by plating on agar medium and an increase of the population of respiring bacteria to 85–95% of the total population. In a separate experiment it was shown that after more than six months of starvation at 15°C, a few of the starved A. salmonicida cells were able to regrow in liquid media after addition of nutrients, but not on agar media. These cells evade detection by direct microscopic respiratory measurement but appear to be reactivated after addition of nutrients.     

Effect of iron on expression of superoxide dismutase by Aeromonas salmonicida and associated resistance to superoxide anion

Abstract Superoxide dismutase activity was detected in Aeromonas salmonicida under iron-replete and iron-limited culture conditions. Under iron-replete conditions an iron superoxide dismutase, molecular mass 50,400 Da, was identified based on inhibition by hydrogen peroxide but not by millimolar concentrations of cyanide. When the available iron in the culture medium was limited by addition of the non-assimilable iron chelator 2,2-dipyridyl, a manganese superoxide dismutase, molecular mass 45,600 Da, was identified, which was resistant to inhibition by either hydrogen peroxide or cyanide. The change in enzyme production would appear to be iron dependent, as addition of FeCl₃ in excess to iron-limited broths resulted in only the iron superoxide dismutase being synthesised. Examination of the location of the superoxide dismutase enzymes revealed that the manganese superoxide dismutase expressed under iron limitation is located in the periplasm, while the iron superoxide dismutase has a cytoplasmic location. The periplasmic manganese superoxide dismutase was able to protect A. salmonicida against extracellular riboflavin-generated superoxide, with A. salmonicida grown under iron-limited conditions exhibiting a 32-fold increase in minimum bactericidal concentration of riboflavin compared to cells cultured under iron-replete conditions. Furthermore, in a time-course study of bactericidal activity of exogenously generated superoxide against A. salmonicida , bacteria grown under iron-replete conditions and expressing cytoplasmic iron superoxide dismutase were rapidly killed, whilst those grown under iron limitation expressing periplasmic manganese superoxide dismutase survived for the duration of the experiment.     

Uptake of the fish pathogen, Aeromonas salmonicida, by rainbow trout (Salmo gairdneri L.)

Aeromonas salmonicida was detected in the blood, kidney and spleen of rainbow trout within 2 min of immersion in a suspension containing 10⁴ cells/ml. Uptake into fish was enhanced by culturing the pathogen in low levels of nutrient, i.e., 0.1% (w/v) brain heart infusion (BHI) broth and by the addition of latex particles to the bacterial suspensions. However, there was no apparent difference in the uptake of pathogenic or non-pathogenic isolates. Moreover, the fish did not succumb to clinical signs of disease.     

叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析

为明确叉角厉蝽Eocanthecona furcellata (Wolff)丝氨酸蛋白酶基因EfSP1及抑制剂基因EfSPI20的基因序列特征和时空转录特征,为其生理功能研究奠定基础。利用PCR克隆技术获得叉角厉蝽唾液腺EfSPI20和EfSP1的完整开放阅读框(Open reading frame, ORF)序列,使用生物信息学软件进行序列分析以及系统进化分析,采用实时荧光定量PCR (Real time quantitativate PCR,RT-qPCR)分析两个基因分别在叉角厉蝽不同发育时期和组织中的表达特征。结果表明,EfSPI20与EfSP1基因完整开放阅读框长度分别为378 bp和921 bp,分别编码125个氨基酸和306个氨基酸,预测均为亲水蛋白质,理论分子量分别为13.48 kDa和33.82 kDa,等电点分别为6.68和5.80,分别有30个和23个氨基酸残基的信号肽序列,EfSPI20有跨膜结构域,EfSP1无跨膜结构域。序列比对显示叉角厉蝽EfSPI20与茶翅蝽Halyomorpha halys PPI同源性最高,氨基酸序列一致性达58%;EfSP1与稻绿蝽Nezara viridula SP同源性最高,氨基酸序列一致性达66%;系统发育树显示叉角厉蝽与同为蝽科的茶翅蝽和稻绿蝽物种亲缘关系近。EfSPI20基因在雌雄成虫和唾液腺中高表达,推测EfSPI20可能具有抑制胰蛋白酶活性的功能和与叉角厉蝽的捕食消化相关;EfSP1基因在卵期、卵巢和肠道中高表达,推测EfSP1可能与叉角厉蝽的生殖功能和蛋白消化相关。     

长江江豚杀鲑气单胞菌的分离鉴定及生物学特性分析

本文通过细菌的分离培养,首次从临床症状表现为溃疡、腐烂的患病江豚表皮分离到一株杀鲑气单胞菌XJ-JT株。通过细菌理化性质鉴定、遗传进化分析、药敏试验、致病性试验对其生物学特性进行分析,结果表明：菌株XJ-JT为革兰氏阴性短杆菌,两端钝圆,无芽孢;细菌分离鉴定的结果显示分离菌株为杀鲑气单胞菌;系统进化分析揭示其基因序列与银鲫源性杀鲑气单胞菌分离株高度同源;20种抗生素的药敏试验结果表明分离菌株对阿米卡星、克拉霉素、克林霉素等8种药物敏感,对头孢噻肟、头孢曲松、卡那霉素中介,对阿莫西林、氨苄西林、四环素等9种药物耐药;人工感染试验,结果显示分离菌对鲫鱼有较强的致病性。