Photobacterium pectinilyticum sp. nov., a novel bacterium isolated from surface seawater of Qingdao offshore.

A Gram-staining-negative, facultative aerobic, motile strain, designated strain ZSDE20T, was isolated from the surface seawater of Qingdao offshore. Phylogenetic analysis of the 16S rRNA gene of strain ZSDE20T, affiliated it to the genus Photobacterium. It was closely related to Photobacterium lutimaris DF-42 T (98.92% 16S rRNA gene sequence similarity). Growth occurred at 4-28ºC (optimum 28ºC), pH 1.0-7.0 (optimum 7.0) and in the presence of 1-7% (w/v) NaCl (optimum 3%). The dominant fatty acids were summed feature 3 (C16:1 ω7c or/and C16:1 ω6c, 34.23%), summed feature 8 (C18:1 ω7c and C18:1 ω6c, 10.36%) and C16:0 (20.05%). The polar lipids of strain ZSDE20T comprised phosphatidylethanolamine, phosphatidylcholine, lyso-phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol dimannoside, phosphatidylinositol mannosides and two unknown lipids. The major respiratory quinone was ubiquinone-8 (Q-8). The DNA G + C content of strain ZSDE20T was 45.6 mol%. Average nucleotide identity (ANI) values between ZSDE20T and its reference species were lower than the threshold for species delineation (95-96%); in silico DNA-DNA hybridization further showed that strain ZSDE20T had less than 70% similarity to its relatives. Based on the polyphasic evidences, strain ZSDE20T is proposed as representing a novel species of the genus Photobacterium, for which the name Photobacterium pectinilyticum sp. nov. is proposed. The type strain is ZSDE20T (= MCCC 1K06283T = KCTC 82885 T).

Functional characterization of NK-lysin in golden pompano Trachinotus ovatus (Linnaeus 1758).

NK-lysin is an important part of the innate immune defence system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, NK-lysin from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length NK-lysin cDNA was 731 bp, which comprised a 5'-UTR of 63 bp, an ORF of 444 bp, and a 3'-UTR of 224 bp, and encoded 147 amino acids; NK-lysin consisted of a conserved saposin B domain and six conserved cysteines that formed three pairs of disulfide bonds. The genomic organization of NK-lysin was also determined and the gene consisted of four introns and five exons. The predicted promoter region of ToNK-lysin contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that ToNK-lysin was ubiquitously expressed in all examined tissues; the highest mRNA levels were observed in the skin, kidney and intestine, while the lowest expression level was detected in the stomach. After P. damselae stimulation, the expression level of NK-lysin mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant NK-lysin expressed in pGEX-6P-1 was approximately 37 kDa. The purified recombinant protein showed antibacterial activity against gram-positive and gram-negative bacteria. The results indicate that golden pompano NK-lysin has potential antimicrobial roles in fish innate immunity.

The intracellular signaling pathway of octopamine upregulating immune resistance functions in Penaeus monodon.

Octopamine (OA), a biogenic monoamine, is known to mediate several immune responses. This study analyzed the effects of OA on immunological regulation in the tiger shrimp Penaeus monodon. The immune parameters including total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and phagocytic activity and clearance efficiency in response to the pathogen, Photobacterium damselae, were determined when shrimp were individually injected with saline or OA at 100 or 1000 pmol shrimp-1. In addition, the intracellular second messengers in haemocyte such as Ca2+ and adenosine 3',5'-cyclic monophosphate (cAMP) were examined in shrimp receiving saline or OA at 1 or 10 nmol shrimp-1. Results showed that all of the immune parameters significantly increased at 2-4 h in OA-injected shrimp except hyaline cells in 100 pmol shrimp-1-injected shrimp at 4 h, but phenoloxidase activity per granulocyte significantly decreased at 2-4 h. However, these had returned to saline control levels after receiving OA for 8 h except differential haemocyte count and phenoloxidase activity per granulocyte for 16 h. An injection of OA also significantly increased the survival rate of shrimp challenged with Pho. damselae. Shrimp receiving OA at 1 and 10 nmol shrimp-1 significantly increased the intracellular Ca2+ concentration ([Ca2+]i) at 30-60 min and 30 min, and cAMP concentration [cAMP]i) at 5-15 min and 15 min, respectively. However, [Ca2+]i at 50-60 min, and [cAMP]i at 30-60 min returned to saline control when the shrimp received OA at 10 nmol shrimp-1, and at 1 and 10 nmol shrimp-1, respectively. These results suggest that OA administration by injection at ≤1000 pmol shrimp-1 mediates transient upregulation of immunity together with the increased resistance of P. monodon to Pho. damselae, which are modulated through intracellular Ca2+ and cAMP second messenger pathways.

Molecular cloning of IL-6, IL-10, IL-11, IFN-ɤ and modulation of pro- and anti-inflammatory cytokines in cobia (Rachycentron canadum) after Photobacterium damselae subsp. piscicida infection.

Photobacterium damselae subsp. piscicida (P. damselae subsp. piscicida) is the agent of Photobacteriosis, a serious fish disease that produces an acute infection and high mortality in farmed cobia. It has been proved that regulation of pro- and anti-inflammatory cytokines play a central role in initiation of proper inflammatory responses against bacterial infection. Here we have analyzed the expression of pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8 and IFN-ɤ) and anti-inflammatory cytokines (IL-10 and IL-11) in spleen and head kidney during acute P. damselae subsp. piscicida infection of cobia. Our data revealed that cytokines tested showed distinct patterns of expression. While TNF-α and IL-8 showed a decay pattern of expression, IL-1ß response was quite late. Moreover, P. damselae subsp. piscicida infection induced the simultaneous expressions of pro-inflammatory (IL-6, IFN-ɤ) and anti-inflammatory (IL-10, IL-11) cytokines. Together these results indicate the innate immunity of cobia is actively suppressed by P. damselae subsp. piscicida.

A proteomic analysis of the iron response of Photobacterium damselae subsp. damselae reveals metabolic adaptations to iron levels changes and novel potential virulence factors.

Photobacterium damselae subsp. damselae (Pdd) is a marine bacterium that can infect numerous species of marine fish as well as other species including humans. Low iron availability is one of the signs that bacterial pathogens can detect in order to begin colonizing their host, and the reduction of iron levels is a nonspecific host defense strategy that prevents bacterial proliferation. In this work a proteomic approach was used to study the gene expression adaptations of a Pdd strain in response to iron availability. A comparative analysis of induced proteins in both high- and low-iron conditions showed profound cellular metabolic adaptations that result, for instance, in amino acid requirement. It also provided important information about the changes that occur in the energetic metabolism induced by the surrounding iron levels, allowing for the identification of novel potential virulence factors. Among others, genes involved in the synthesis and transport of a vibrioferrin-like siderophore were identified for the first time. In addition to plasmid pPHDD1-encoded Dly and HlyA hemolysins, a pPHDD1-borne operon, which may encode a transferrin receptor, was also found. This operon identification suggests that this virulence plasmid could encode so-far unknown additional virulence factors other than hemolysins.

Thioredoxin of golden pompano involved in the immune response to Photobacterium damselae.

Thioredoxin (TRX) is one of the key systems responsible for keeping the intracellular environment in a highly reduced state. In this study, a full-length TRX cDNA sequence (ToTRX) from golden pompano Trachinotus ovatus was identified after pyrosequencing of golden pompano cDNA library. ToTRX cDNA is comprised of 786 bp, and contained a 324 bp open reading frame (ORF) encoding a 107 amino acid polypeptide, a 5' untranslated region (UTR) of 116 bp, and a long 3'- UTR of 346 bp. Multiple sequence alignment revealed that ToTRX contained the highly conserved redox active disulphide/dithiol site (CGPC) of the thioredoxin active family, and phylogenetic tree showed that ToTRX had a closer evolution relationship with TRX from Oplegnathus fasciatus and Anoplopoma fimbria. ToTRX mRNA is ubiquitously expressed in all detected tissues with the higher expression levels in the stomach, gill and fin tissues. The expression of ToTRX mRNA was significantly up-regulated in liver, kidney, intestine and spleen of golden pompano injected with Photobacterium damselae. The recombinant ToTRX protein (rToTRX) was expressed in Escherichia coli BL21 (DE3), and then purified and refolded. The insulin disulfides assay was performed to investigate the enzymatic oxidoreductase activity of rToTRX, and the results demonstrated that rToTRX exhibited a high reducing activity in presence of DTT, while no activity was observed in the regroup without DTT and blank control group. Over all, the study provided the useful information to help further understand the functional mechanism of TRX in marine fish immunity.

Photobacterium panuliri sp. nov., an alkalitolerant marine bacterium isolated from eggs of spiny lobster, Panulirus penicillatus from Andaman Sea.

A facultative anaerobe, alkalitolerant, gram-negative marine bacterium strain LBS5(T), was isolated from eggs carried on the pleopods of female spiny lobster (Panulirus penicillatus) in Andaman Sea from a depth of 3.5 m. Heterotrophic growth was observed at 15-38 °C and pH 5.5-11. Optimum growth occurred at 28 °C and pH 7.5. It can grow in the presence of 0.5-7 % NaCl (w/v), and the optimal NaCl required for growth was 2-4 %. 16S rRNA gene sequence analysis revealed the strain LBS5(T) belongs to the genus Photobacterium and showed 99.6 % similarity with P. aquae AE6(T), 98.2 % with P. aphoticum M46(T), 97 % with P. rosenbergii CC1(T), 96.9 % with P. lutimaris DF-42(T), and 96.6 % with P. halotolerans MACL01(T). The DNA-DNA similarities between strains LBS5(T) with other closely related strains were well below 70 %. The DNA G + C content was 50.52 (±0.9) mol%. The major fatty acids were C16:1w7c/w6c, C18:1w6c/w7c, C16:0, C15:0 iso, C16:0 10-methyl/17:1 iso w9c, C17:0 iso. Polar lipids included a phosphatidylglycerol, a diphosphatidylglycerol, a phosphatidylethanolamine, and one unidentified lipid. Based on the polyphasic evidences, strain LBS5(T) represents a novel species of the genus Photobacterium for which Photobacterium panuliri sp. nov. is proposed. The type strain is LBS5(T) (=DSM 27646(T) = LMG 27617(T) = JCM 19199(T)).

Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida.

Abstract Cobalamin (vitamin B(12)) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B(12). A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B(12)-binding protein precursor of P. profundum, 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida, but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida, the recombinant protein was expressed with a C-terminal His(6)-tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 +/- 2 microm.

Invasion and survival of Photobacterium damselae subsp. piscicida in non-phagocytic cells of gilthead sea bream, Sparus aurata L.

Fluorescence microscopy and gentamicin protection assays were used to investigate the ability of four Photobacterium damselae subsp. pisicida (Phdp) strains to adhere to and to invade the fish epithelial cell line, SAF-1, derived from Sparus aurata. All strains tested were detected intracellularly using both techniques, although internalization levels varied among strains. Treatment with cytochalasin D and experiments carried out at 4 degrees C demonstrated that a functional host cell cytoskeleton and active cell metabolism are necessary for bacterial internalization. Intracellular bacteria were detected for up to 7 days with a round morphology and were stained with DAPI, indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that Phdp are capable of adhering, entering and surviving within the non-phagocytic epithelial cell line SAF-1, which may be important for persistence and establishment of a carrier state in S. aurata.

Catabolite regulation of enzymatic activities in a white pox pathogen and commensal bacteria during growth on mucus polymers from the coral Acropora palmata.

Colonization of host mucus surfaces is one of the first steps in the establishment of coral-associated microbial communities. Coral mucus contains a sulfated glycoprotein (in which oligosaccharide decorations are connected to the polypeptide backbone by a mannose residue) and molecules that result from its degradation. Mucus is utilized as a growth substrate by commensal and pathogenic organisms. Two representative coral commensals, Photobacterium mandapamensis and Halomonas meridiana, differed from a white pox pathogen Serratia marcescens PDL100 in the pattern with which they utilized mucus polymers of Acropora palmata. Incubation with the mucus polymer increased mannopyranosidase activity in S. marcescens, suggestive of its ability to cleave off oligosaccharide side chains. With the exception of glucosidase and N-acetyl galactosaminidase, glycosidases in S. marcescens were subject to catabolite regulation by galactose, glucose, arabinose, mannose and N-acetyl-glucosamine. In commensal P. mandapamensis, at least 10 glycosidases were modestly induced during incubation on coral mucus. Galactose, arabinose, mannose, but not glucose or N-acetyl-glucosamine had a repressive effect on glycosidases in P. mandapamensis. Incubation with the mucus polymers upregulated 3 enzymatic activities in H. meridiana; glucose and galactose appear to be the preferred carbon source in this bacterium. Although all these bacteria were capable of producing the same glycosidases, the differences in the preferred carbon sources and patterns of enzymatic activities induced during growth on the mucus polymer in the presence of these carbon sources suggest that to establish themselves within the coral mucus surface layer commensals and pathogens rely on different enzymatic activities.

Observations on the estrogenic activity and concentration of 17beta-estradiol in the discharges of 12 wastewater treatment plants in southern australia.

There is very little information on the overall level of estrogenic activity, or concentrations of specific hormonal compounds in wastewater treatment plant (WWTP) discharges in Australia, compared with Europe, Japan, and North America. To partly address this, in 2004, water samples were collected as "grab" or "spot" samples from 12 WWTP facilities across southern Victoria at the point at which effluent enters the environment, either as recycled water or direct discharge to the receiving water. The WWTPs were of a variety of treatment types and served a diverse range of rural and regional municipalities. For instance, of the 12 WWTPs, 3 served municipalities with populations greater than 100,000, 4 with populations between 20,000 and 100,000, and 5 with populations less than 5,000. The principal treatment process in six was an activated sludge system, and three were trickle-filter-based systems. The remaining plants fall into a "miscellaneous" category, each plant having a mixture of treatment processes within the overall systems. The estrogenic activity and 17beta-estradiol concentrations of the samples were assessed using a yeast-based, in vitro reporter gene assay and enzyme-linked immunosorbant assays, respectively. Most of the effluents showed estrogenic activity in the assays (hER, no response: 7.9 ng/L EEQ; mER, no response: 44.5 ng/L EEQ). There was no correlation between estrogenic response and the results of a concurrent toxicity assay, suggesting that a lack of bioassay response was related to lack of estrogenic compounds, rather than the direct toxic effect of the sample. Estradiol concentrations were for the most part in the range 2-5 ng/L, with one sample at 18 ng/L. Despite the assurance our results might provide (of minimal impact in most cases if there is significant dilution), there is still a need for further extensive on-ground reassurance research to provide data for higher-level risk assessment by industry and government agencies. In particular, more research is warranted to verify the estrogenic activity and to expand the range of specific hormone/metabolites reported in these studies. Moreover, studies are required to determine if the estrogenic activity reported in this and other recent Australian studies is sufficient to induce a physiological effect in exposed aquatic organisms, especially Australian native fish.

Molecular cloning and expression analysis of sea bass (Dicentrarchus labrax L.) tumor necrosis factor-alpha (TNF-alpha).

In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.

Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits.

Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

Invasion of fish epithelial cells by Photobacterium damselae subsp. piscicida: evidence for receptor specificity, and effect of capsule and serum.

Photobacterium damselae subsp. piscicida is a fish pathogen which causes serious disease in commercial warmwater fish species. Because information on the initial stages of the infection is scarce, an investigation of the invasion ability of this pathogen was undertaken utilizing a fish epithelial cell line (epithelioma papillosum carpio, EPC), a virulent capsulated strain of P. damselae (MT1415), an avirulent non-capsulated strain of P. damselae (EPOY-8803-ii) and Escherichia coli HB101 as a non-invasive control. P. damselae was found to be able to adhere to and invade fish epithelial cells and remain inside them for 6-9 h. There were no significant differences in invasiveness between the capsulated and non-capsulated strains. A kinetics study demonstrated that P. damselae invasiveness was more efficient at low m.o.i., reaching saturation at higher m.o.i., suggesting internalization may be receptor-mediated. Invasion efficiency (IE) was significantly higher than in the control E. coli HB101. Engulfment of bacteria was possibly by an endocytic process and was unaffected by killing the bacteria with UV light. However, heat-killed bacteria had significantly reduced invasion capability. Ultrastructural studies showed that inside the epithelial cells, the bacteria remained within large vacuoles for a few hours and no evidence of intracellular replication was found, by either fluorescence or electron microscopic studies. Normal sea bass serum slightly reduced the invasion capability of the MT1415 strain, but heat-inactivated normal serum had no effect. On the other hand, heat-inactivated fish antiserum raised against the same strain reduced the percentage of invaded epithelial cells by 50%. As for other pathogens, an intracellular phase of P. damselae may be a mechanism to delay or avoid phagocytosis and host immune responses, favouring the spread of infection.

Microbial adaptations to the psychrosphere/piezosphere.

Low temperature and high pressure deep-sea environments occupy the largest fraction of the biosphere. Nevertheless, the molecular adaptations that enable life to exist under these conditions remain poorly understood. This article will provide an overview of the current picture on high pressure adaptation in cold oceanic environments, with an emphasis on genetic experiments performed on Photobacterium profundum. Thus far genes which have been found or implicated as important for pressure-sensing or pressure-adaptation include genes required for fatty acid unsaturation, the membrane protein genes toxR and rseC and the DNA recombination gene recD. Many deep-sea bacteria possess genes for the production of omega-3 polyunsaturated fatty acids. These could be of biotechnological significance since these fatty acids reduce the risk of cardiovascular disease and certain cancers and are useful as dietary supplements.