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The molecular mechanisms by which deep-sea bacteria such as Photobacterium profundum SS9 grow optimally at high pressure and cold temperatures are poorly understood. In this thesis, four previously identified P. profundum SS9R mini-transposon mutants were investigated. The high pressure-sensitive FL31 mutant has an insertion in a putative rctB gene. V. cholerae RctB protein is essential for replication initiation at the origin of chromosome II, oriCII. In this thesis, the potential link between pressure sensitivity, RctB and oriCII was investigated using a plasmid-based system in E. coli. The P. profundum oriCII was identified and the incompatibility region (incII) was shown to inhibit oriCII activity. The putative rctB gene was essential for oriCII function and when truncated, as in FL31, it conferred insensitivity to incII inhibition, indicating a link between downregulation of oriCII activity and survival at increased pressure. These findings provide the first characterisation of the replication origin of chromosome II in an organism other than V. cholerae and point to a critical control mechanism for chromosome duplication in response to changes in a key environmental factor, pressure. The FL24, FL25 and FL26 mutants display a cold-sensitive phenotype only on agar. In this study FL25 and FL26 were found to lack smooth LPS and displayed increased swimming motility compared to the parent, which could indicate alterations in their polar flagellum function. Since the polar flagellum can act as a surface sensor that induces changes in gene expression, the cold-sensitive colony growth phenotypes of the S-LPS mutants could stem from defects in this surface sensing mechanism. The observed lack of polar flagellum in FL24 rendered the mutant non-motile but was not directly responsible for its cold-sensitive phenotype. Hypothetically, the cold sensitivity of FL24 could stem from the transposon insertion in gene encoding the FliS chaperone and subsequent intracellular accumulation of flagellin.

The mechanism of high pressure-adapted growth in the deep-sea bacterium Photobacterium profundum SS9 is poorly understood. To gain further insights, two P. profundum SS9R mutants were investigated. FL23 (pbpra3229::m-Tn10) and FL28 (pbpra1039::m-Tn10) had been previously characterised as high pressuresensitive and pressure-enhanced, respectively. FL23 had a growth defect at atmospheric pressure but failed to show high pressure-adapted growth on solid agar. Pbpra3229 is 75 % identical to E. coli DiaA (stimulator of DNA replication and critical for the timely initiation of replication) and 45% identical to E. coli GmhA (essential for lipopolysaccharide core biosynthesis), which led to an investigation into whether either process was affected in FL23. However, the lipopolysaccharide of FL23 and its parent strain were identical, which suggests that Pbpra3229 is not a GmhA homologue. In contrast, the pbpra3229 and E. coli diaA genes were functionally interchangeable and both restored the timing of DNA replication in an E. coli diaA mutant. FL28 had growth and morphological defects at high pressure, but both phenotypes were exacerbated at atmospheric pressure. Pbpra1039 is 55% identical to E. coli SeqA, which is a negative regulator of DNA replication and also essential for timely initiation. Pbpra1039 was shown to be a functional homologue of E. coli SeqA, as pbpra1039 partially complemented the DNA replication defect of an E. coli seqA mutant. Combined, these findings provide evidence that Pbpra3229 is a DiaA homologue, whereas Pbpra1039 is a cold adapted SeqA homologue, and that both positive and negative regulation of initiation of DNA replication are essential for the ability of P. profundum SS9 to adapt to deep-sea conditions. A marine metagenomic library was also screened for clones that produced novel cell envelope polysaccharides and tools were developed to identify cell envelope polysaccharides in P. profundum SS9.

Abstract Farming of cobia (Rachycentron canadum) is one of the important maricultured fish species in Taiwan. However, bacterial diseases have plagued cobia aquaculture industry. In this study, based on the growth characteristic, morphological, and biochemical properties of an isolated bacterium, from diseased fish of were similar to those of Photobacterium damselae subsp. piscicida. The 16S rRNA sequence showed 99% identity with P. damselae subsp. Piscicida (GenBank accession number AY147860.1). The pathogenicity experiments were tested by intraperitoneal injection (IP) in juvenile cobia (12~17 g). The LD50 value of P. damselae subsp. piscicida for cobia was 1.0¡Ñ105.6 cells/ml. In order to understand the spreading routes of P. damselae subsp. piscicida in cobia, we infected in cobia fries with P. damselae subsp. piscicida. The results showed that 6h postinfection, P. damselae subsp. piscicida was isolated from gills and gastrointestines. And at 96h P. damselae subsp. piscicida could be isolated form all organs. Histology and fluorescence in situ hybridization showed that the bacterium enters via gills or gastrointestinal tracts and spreaded to kidney and liver, and eventually developed into systemic infection. In addition, genus-specific sequence of 16S rRNA of pasteurellosis was amplified by PCR using the PDPF and PFPR primer. A 847 bp fragment was observed in all the nodules organ and bacterial suspension. The technique is capable of rapid identification of the sequence pathogen in fish.

The purpose of this study was for building the data on blood and serum chemistry of grouper ‚ and expect these data can be helpful to know the fish condition in early stages‚ in order to remedy as soon as possible and decrease the loss that caused of fish disease. Three experiments were conducted to establish the data on blood and serum chemistry of grouper. In the first experiment‚ the clinical chemistry including red and white blood cell counts‚ Glutamic oxalacetic transaminase (GOT)‚ Glutamic pyruvic transaminase (GPT)‚ total protein‚ albumin and blood ion of Na‚ K and Ca were built in different size of healthy orange spotted grouper¡]Epinephelus coioides¡^. The results showed that the white blood cell counts was divided into two major parts 70-90&#x00B4;103 cell/ml and 110-140&#x00B4;103 cell/ml. Red blood cell counts was landed on 1.85-3.21&#x00B4;106 cell/ml. The range of GOT and GPT were 0-45 IU/L and 0-350 IU/L. Total protein and albumin were 3.8-5.6 and 0.7-1.6 g/dL. In the second experiment‚ the clinical chemistry of orange spotted grouper after infected by dactylogurus‚ trichodinla‚ nervous necrosis virus (NNV) and NNV with Amyloodinium ocellatum were collected and compared with normal orange spotted grouper. The white blood cell counts and the concentration of K+ in the infected NNV and dactylogurus of fish were significantly higher (P<0.05) than normal fish‚ while the granulocyte in infected fish was significantly lower (P<0.05) than normal fish. In the dactylogurus and trichodinla infections of fish‚ the concentration of Ca2+ was significantly higher (P<0.05) than normal fish. In the third experiment‚ the size of monocytes and neutrophils of brown marbled grouper¡]E. fuscoguttatus¡^ were grown in 8 hours and 16 hours‚ respectively‚ after challenged with Photobacterium damselae subsp. damselae‚ and the eosinophils size of challenge group fish bigger (P<0.05) than control group fish after challenged for 16 hours.

Clean and spiked sediment formulations of various silt sand and clay sand ratios were tested for toxicity using a bioassay that utilizes bioluminescent bacteria Measured toxicities of clean and copper sulfate–spiked sediments were negatively but nonlinearly related with percent silt and percent clay, but no significant relationship existed between measured toxicity and sediment composition for methyl parathion–spiked formulations Results suggest that solid phase sediment bioassays using bioluminescent bacteria may be useful for testing the toxicities of single contaminants in formulated artificial sediments of known particle size composition, and for repeated samples collected from the same site However, extreme caution must be taken when testing sediments of varying composition or which may be differentially contaminated or contain a suite of contaminants.

Bdellovibrionaceae (BD) are unique, predatory, endoparasitic, Gram-negative bacteria. As the world's smallest living hunter they prey on other Gram-negative bacteria giving them potential as biological control agents. Prior to this study, however, there were no reports of BD in New Zealand. The overall aim of this research was to isolate BD from New Zealand sources, characterise them and investigate their potential role as a biological control agent. The history, characteristics, life cycle and mechanism of predation of this organism are reviewed and the possibility of the industrial applications of BD, are discussed. In this study, a halophilic species of BD was isolated from fourteen coastal sea water sites around New Zealand. Thirteen isolates were characterised using proven characterisation techniques including general, microscopic and molecular techniques. It was found that the isolates were taxonomically identical or very closely related to each other and belong to the genus Bacteriovorax. The predation pattern of BD isolates was examined against a group of Gram negative bacteria in solid and liquid media. The predation patterns and efficiencies of the different BD isolates were similar, which confirms that the BD isolates are closely related, are selective in their predation, and prey on some Gram-negative bacteria but not all. The rapid loss of culture viability of BD is well known, but no studies have been reported to date on the survival of pure cultures of BD at different temperatures. The survival rate of BD in dense suspensions at different temperatures without host bacteria was investigated and it was observed that pure BD cultures can be stored with minimal reduction in numbers at temperatures ranging from 4°C to 20°C. However, significant reductions in numbers were observed at -1 8"C, 30°C and 37°C after 13 to 16 days. The effects of the 13 New Zealand BD isolates on the growth of a population of Photobacterium phosphoreum were examined to select the best isolate for in vitro application. All of the isolates tested had considerable reduction effect against P. phosphoreum. Some isolates were more effective than others, despite their taxonomic similarity to each other. The isolate OT2 was selected for further studies based on these results. The in vitro efficacy of BD was assessed against late exponential cultures of a seafood spoilage bacterium, P. phosphoreum, originally isolated from Cod fillets from Denmark. Loglo reductions of P. phosphoreum and some other Gram-negative bacteria ranged from 4.5 to 4.8 after 9 h of incubation at 25OC. BD was effective in reducing the numbers of P. phosphoreum at pH 5.5 to 8.5 and salinity 0.9 to 4.5% (wlv). A significant interaction was observed between the prey and predator concentrations and nutrient concentration. Prey concentrations were observed to be the most vital factor in predation and the most favourable predation conditions were at a prey concentration of -8 loglo colony forming units (CFU)/mL, together with a predator concentration of 3 - 7 loglo plaque forming units (PFU)/mL and a prey : predator ratio of >5.0. The thresholds of the prey and predator concentrations for predation were observed to be 3.7 loglo CFUImL and 3.9 loglo PFUImL, respectively. The trials carried out in this study focused on the efficiency of BD on a pure culture of one organism, P. phosphoreum and not on mixed cultures of Gramnegative spoilage bacteria, the normal condition observed in saltwater fish. There has been very little research in this field and the results of these trials suggest further investigation into the effect of BD on mixed cultures of Gram-negative spoilage organisms is warranted. Since only one isolate of BD (OT2) was examined against only one spoilage bacterium (P. phosphoreum) in liquid medium, the evidence of these findings must be restricted to these particular conditions. Future studies, using a range of BD isolates against a mixture of spoilage and pathogenic organisms in solid medium are warranted. The biopreservation capability of BD in extending the shelf life of king salmon was evaluated. A significant effect was observed at 20°C but not at 10°C. At 20°C the shelf life was extended through extension of the lag phase of growth of the prey bacteria and a reduction in total numbers attained. Sensory evaluation of the salmon product being tested confirmed that the shelf life was extended. However, at 10°C there was no reduction in prey organisms, which suggested that the strain of BD used is ineffective at refrigeration temperatures.

Bdellovibrionaceae (BD) are unique, predatory, endoparasitic, Gram-negative bacteria. As the world's smallest living hunter they prey on other Gram-negative bacteria giving them potential as biological control agents. Prior to this study, however, there were no reports of BD in New Zealand. The overall aim of this research was to isolate BD from New Zealand sources, characterise them and investigate their potential role as a biological control agent. The history, characteristics, life cycle and mechanism of predation of this organism are reviewed and the possibility of the industrial applications of BD, are discussed. In this study, a halophilic species of BD was isolated from fourteen coastal sea water sites around New Zealand. Thirteen isolates were characterised using proven characterisation techniques including general, microscopic and molecular techniques. It was found that the isolates were taxonomically identical or very closely related to each other and belong to the genus Bacteriovorax. The predation pattern of BD isolates was examined against a group of Gram negative bacteria in solid and liquid media. The predation patterns and efficiencies of the different BD isolates were similar, which confirms that the BD isolates are closely related, are selective in their predation, and prey on some Gram-negative bacteria but not all. The rapid loss of culture viability of BD is well known, but no studies have been reported to date on the survival of pure cultures of BD at different temperatures. The survival rate of BD in dense suspensions at different temperatures without host bacteria was investigated and it was observed that pure BD cultures can be stored with minimal reduction in numbers at temperatures ranging from 4°C to 20°C. However, significant reductions in numbers were observed at -1 8"C, 30°C and 37°C after 13 to 16 days. The effects of the 13 New Zealand BD isolates on the growth of a population of Photobacterium phosphoreum were examined to select the best isolate for in vitro application. All of the isolates tested had considerable reduction effect against P. phosphoreum. Some isolates were more effective than others, despite their taxonomic similarity to each other. The isolate OT2 was selected for further studies based on these results. The in vitro efficacy of BD was assessed against late exponential cultures of a seafood spoilage bacterium, P. phosphoreum, originally isolated from Cod fillets from Denmark. Loglo reductions of P. phosphoreum and some other Gram-negative bacteria ranged from 4.5 to 4.8 after 9 h of incubation at 25OC. BD was effective in reducing the numbers of P. phosphoreum at pH 5.5 to 8.5 and salinity 0.9 to 4.5% (wlv). A significant interaction was observed between the prey and predator concentrations and nutrient concentration. Prey concentrations were observed to be the most vital factor in predation and the most favourable predation conditions were at a prey concentration of -8 loglo colony forming units (CFU)/mL, together with a predator concentration of 3 - 7 loglo plaque forming units (PFU)/mL and a prey : predator ratio of >5.0. The thresholds of the prey and predator concentrations for predation were observed to be 3.7 loglo CFUImL and 3.9 loglo PFUImL, respectively. The trials carried out in this study focused on the efficiency of BD on a pure culture of one organism, P. phosphoreum and not on mixed cultures of Gramnegative spoilage bacteria, the normal condition observed in saltwater fish. There has been very little research in this field and the results of these trials suggest further investigation into the effect of BD on mixed cultures of Gram-negative spoilage organisms is warranted. Since only one isolate of BD (OT2) was examined against only one spoilage bacterium (P. phosphoreum) in liquid medium, the evidence of these findings must be restricted to these particular conditions. Future studies, using a range of BD isolates against a mixture of spoilage and pathogenic organisms in solid medium are warranted. The biopreservation capability of BD in extending the shelf life of king salmon was evaluated. A significant effect was observed at 20°C but not at 10°C. At 20°C the shelf life was extended through extension of the lag phase of growth of the prey bacteria and a reduction in total numbers attained. Sensory evaluation of the salmon product being tested confirmed that the shelf life was extended. However, at 10°C there was no reduction in prey organisms, which suggested that the strain of BD used is ineffective at refrigeration temperatures.

Bdellovibrionaceae (BD) are unique, predatory, endoparasitic, Gram-negative bacteria. As the world's smallest living hunter they prey on other Gram-negative bacteria giving them potential as biological control agents. Prior to this study, however, there were no reports of BD in New Zealand. The overall aim of this research was to isolate BD from New Zealand sources, characterise them and investigate their potential role as a biological control agent. The history, characteristics, life cycle and mechanism of predation of this organism are reviewed and the possibility of the industrial applications of BD, are discussed. In this study, a halophilic species of BD was isolated from fourteen coastal sea water sites around New Zealand. Thirteen isolates were characterised using proven characterisation techniques including general, microscopic and molecular techniques. It was found that the isolates were taxonomically identical or very closely related to each other and belong to the genus Bacteriovorax. The predation pattern of BD isolates was examined against a group of Gram negative bacteria in solid and liquid media. The predation patterns and efficiencies of the different BD isolates were similar, which confirms that the BD isolates are closely related, are selective in their predation, and prey on some Gram-negative bacteria but not all. The rapid loss of culture viability of BD is well known, but no studies have been reported to date on the survival of pure cultures of BD at different temperatures. The survival rate of BD in dense suspensions at different temperatures without host bacteria was investigated and it was observed that pure BD cultures can be stored with minimal reduction in numbers at temperatures ranging from 4°C to 20°C. However, significant reductions in numbers were observed at -1 8"C, 30°C and 37°C after 13 to 16 days. The effects of the 13 New Zealand BD isolates on the growth of a population of Photobacterium phosphoreum were examined to select the best isolate for in vitro application. All of the isolates tested had considerable reduction effect against P. phosphoreum. Some isolates were more effective than others, despite their taxonomic similarity to each other. The isolate OT2 was selected for further studies based on these results. The in vitro efficacy of BD was assessed against late exponential cultures of a seafood spoilage bacterium, P. phosphoreum, originally isolated from Cod fillets from Denmark. Loglo reductions of P. phosphoreum and some other Gram-negative bacteria ranged from 4.5 to 4.8 after 9 h of incubation at 25OC. BD was effective in reducing the numbers of P. phosphoreum at pH 5.5 to 8.5 and salinity 0.9 to 4.5% (wlv). A significant interaction was observed between the prey and predator concentrations and nutrient concentration. Prey concentrations were observed to be the most vital factor in predation and the most favourable predation conditions were at a prey concentration of -8 loglo colony forming units (CFU)/mL, together with a predator concentration of 3 - 7 loglo plaque forming units (PFU)/mL and a prey : predator ratio of >5.0. The thresholds of the prey and predator concentrations for predation were observed to be 3.7 loglo CFUImL and 3.9 loglo PFUImL, respectively. The trials carried out in this study focused on the efficiency of BD on a pure culture of one organism, P. phosphoreum and not on mixed cultures of Gramnegative spoilage bacteria, the normal condition observed in saltwater fish. There has been very little research in this field and the results of these trials suggest further investigation into the effect of BD on mixed cultures of Gram-negative spoilage organisms is warranted. Since only one isolate of BD (OT2) was examined against only one spoilage bacterium (P. phosphoreum) in liquid medium, the evidence of these findings must be restricted to these particular conditions. Future studies, using a range of BD isolates against a mixture of spoilage and pathogenic organisms in solid medium are warranted. The biopreservation capability of BD in extending the shelf life of king salmon was evaluated. A significant effect was observed at 20°C but not at 10°C. At 20°C the shelf life was extended through extension of the lag phase of growth of the prey bacteria and a reduction in total numbers attained. Sensory evaluation of the salmon product being tested confirmed that the shelf life was extended. However, at 10°C there was no reduction in prey organisms, which suggested that the strain of BD used is ineffective at refrigeration temperatures.

Bdellovibrionaceae (BD) are unique, predatory, endoparasitic, Gram-negative bacteria. As the world's smallest living hunter they prey on other Gram-negative bacteria giving them potential as biological control agents. Prior to this study, however, there were no reports of BD in New Zealand. The overall aim of this research was to isolate BD from New Zealand sources, characterise them and investigate their potential role as a biological control agent. The history, characteristics, life cycle and mechanism of predation of this organism are reviewed and the possibility of the industrial applications of BD, are discussed. In this study, a halophilic species of BD was isolated from fourteen coastal sea water sites around New Zealand. Thirteen isolates were characterised using proven characterisation techniques including general, microscopic and molecular techniques. It was found that the isolates were taxonomically identical or very closely related to each other and belong to the genus Bacteriovorax. The predation pattern of BD isolates was examined against a group of Gram negative bacteria in solid and liquid media. The predation patterns and efficiencies of the different BD isolates were similar, which confirms that the BD isolates are closely related, are selective in their predation, and prey on some Gram-negative bacteria but not all. The rapid loss of culture viability of BD is well known, but no studies have been reported to date on the survival of pure cultures of BD at different temperatures. The survival rate of BD in dense suspensions at different temperatures without host bacteria was investigated and it was observed that pure BD cultures can be stored with minimal reduction in numbers at temperatures ranging from 4°C to 20°C. However, significant reductions in numbers were observed at -1 8"C, 30°C and 37°C after 13 to 16 days. The effects of the 13 New Zealand BD isolates on the growth of a population of Photobacterium phosphoreum were examined to select the best isolate for in vitro application. All of the isolates tested had considerable reduction effect against P. phosphoreum. Some isolates were more effective than others, despite their taxonomic similarity to each other. The isolate OT2 was selected for further studies based on these results. The in vitro efficacy of BD was assessed against late exponential cultures of a seafood spoilage bacterium, P. phosphoreum, originally isolated from Cod fillets from Denmark. Loglo reductions of P. phosphoreum and some other Gram-negative bacteria ranged from 4.5 to 4.8 after 9 h of incubation at 25OC. BD was effective in reducing the numbers of P. phosphoreum at pH 5.5 to 8.5 and salinity 0.9 to 4.5% (wlv). A significant interaction was observed between the prey and predator concentrations and nutrient concentration. Prey concentrations were observed to be the most vital factor in predation and the most favourable predation conditions were at a prey concentration of -8 loglo colony forming units (CFU)/mL, together with a predator concentration of 3 - 7 loglo plaque forming units (PFU)/mL and a prey : predator ratio of >5.0. The thresholds of the prey and predator concentrations for predation were observed to be 3.7 loglo CFUImL and 3.9 loglo PFUImL, respectively. The trials carried out in this study focused on the efficiency of BD on a pure culture of one organism, P. phosphoreum and not on mixed cultures of Gramnegative spoilage bacteria, the normal condition observed in saltwater fish. There has been very little research in this field and the results of these trials suggest further investigation into the effect of BD on mixed cultures of Gram-negative spoilage organisms is warranted. Since only one isolate of BD (OT2) was examined against only one spoilage bacterium (P. phosphoreum) in liquid medium, the evidence of these findings must be restricted to these particular conditions. Future studies, using a range of BD isolates against a mixture of spoilage and pathogenic organisms in solid medium are warranted. The biopreservation capability of BD in extending the shelf life of king salmon was evaluated. A significant effect was observed at 20°C but not at 10°C. At 20°C the shelf life was extended through extension of the lag phase of growth of the prey bacteria and a reduction in total numbers attained. Sensory evaluation of the salmon product being tested confirmed that the shelf life was extended. However, at 10°C there was no reduction in prey organisms, which suggested that the strain of BD used is ineffective at refrigeration temperatures.