Influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum).

The influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum), was studied experimentally in the laboratory and at a fish farm. In experiment I, the effect of parasitic infection on columnaris disease was studied using F. columnare carrier fish. The fish were exposed to Diplostomum spathaceum cercariae and a set of other stressors in order to induce clinical columnaris infection. Parasitic infection and other stressors failed to induce the disease. Disease occurred when the fish were challenged with F. columnare, but D. spathaceum infection did not enhance the severity of the infection. In experiment II, the influence of rearing density and water temperature was studied. Overall mortality was highest in fish at normal rearing density with high temperature (+23 degrees C). At low temperature (+18 degrees C) mortality was not affected by rearing density, but the transmission of columnaris disease was faster at normal rearing density at both temperatures. This supports the view that reduction of fish density could be used in prevention of columnaris disease especially if water temperature is high. Because the lower rearing density can also decrease the transmission of ectoparasites and penetrating endoparasites, it could be an efficient tool in ecological disease management.

Effect of Pseudomonas sp. MT5 baths on Flavobacterium columnare infection of rainbow trout and on microbial diversity on fish skin and gills.

Use of Pseudomonas sp. strain MT5 to prevent and treat Flavobacterium columnare infection was studied in 2 experiments with fingerling rainbow trout Oncorhynchus mykiss. In the first experiment, length heterogeneity analysis of PCR-amplified DNA fragments (LH-PCR) was used to assess the effect of antagonistic baths on the microbial diversity of healthy and experimentally infected fish. In the 148 samples studied, no difference was found between bathed and unbathed fish, and 3 fragment lengths were detected most frequently: 500 (in 75.7% of the samples), 523 (62.2%) and 517 bp (40.5%). The species contributing to these fragment sizes were Pseudomonas sp., Rhodococcus sp. and F. columnare, respectively. A specific PCR for detection of Pseudomonas sp. MT5 was designed, but none of the tissue samples were found to be positive, most likely indicating poor adhesion of the strain during bathing. LH-PCR was found to be a more powerful tool for detecting F. columnare in fish tissue than traditional culture methods (chi2 = 3.9, df = 1, p < 0.05). Antagonistic baths had no effect on the outbreak of infection or on fish mortality. F. columnare was also detected in healthy fish prior to and after experimental infection, indicating that these fish were carriers of the disease. In the second experiment, intensive Pseudomonas sp. MT5 antagonistic baths were given daily to rainbow trout suffering from a natural columnaris infection. Again, the antagonistic bacteria had no effect on fish mortality, which reached 95 % in both control and antagonist-treated groups in 7 d.

Production and characterization of the recombinant Sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase.

Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen. In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed. Hence, a two-step purification protocol was developed which allowed obtaining 15-20 mg of rePCP4MO from 1 L culture. The rePCP4MO revealed identity with native enzyme by SDS-PAGE and N-terminal sequence analyses. Furthermore, a polyclonal PCP4MO antibody was produced with GST-PCP4MO and purified by immunoaffinity chromatography, where both the native and recombinant forms of PCP4MO showed interaction. However, rePCP4MO was identified as apoprotein with no evidence for a typical flavoprotein spectrum. The catalytic activity could be detected in the presence of FAD. The K(m) and V(max) values for PCP were 50 microM and 30 nmol/min/mg, respectively.

Degradation of 2,3,4,6-tetrachlorophenol at low temperature and low dioxygen concentrations by phylogenetically different groundwater and bioreactor bacteria.

Effects of low temperature and low oxygen partial pressure on the occurrence and activity of 2,3,4,6-tetrachlorophenol degrading bacteria in a boreal chlorophenol contaminated groundwater and a full-scale fluidized-bed bioreactor were studied using four polychlorophenol degrading bacterial isolates of different phylogenetic backgrounds. These included an alpha-proteobacterial Sphingomonas sp. strain MT1 isolated from the full-scale bioreactor and three isolates from the contaminated groundwater which were identified as beta-proteobacterial Herbaspirillum sp. K1, a Gram-positive bacterium with high G + C content Nocardioides sp. K44 and an alpha-proteobacterial Sphingomonas sp. K74. The Sphingomonas strains K74 and MT1 and Nocardioides sp. K44 degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol as the sole carbon and energy sources. Close to stoichiometric inorganic chloride release with the 2,3,4,6-tetrachlorophenol removal and the absence of methylation products indicated mineralization. Tetrachlorophenol degradation by the Herbaspirillum sp. K1 was enhanced by yeast extract, malate, glutamate, pyruvate, peptone and casitone. At 8 degrees C, Sphingomonas sp. K74 had the highest specific degradation rate (mu(max) = 4.9 x 10(-2) mg h(-1) cell(-1)) for 2,3,4,6-tetrachlorophenol. The Nocardioides strain K44 had the highest affinity (K(s) = 0.46 mg l(-1)) fortetrachlorophenol. K1 and MT1 grew microaerophilically in semisolid glucose medium. Furthermore, the growth of MT1 was inhibited in liquid glucose medium at high oxygen partial pressure indicating sensitivity to accumulating toxic oxygen species. On the other hand, trichlorophenol degradation was not affected by oxygen concentration (2-21%). The isolates K44, K74 and MT1, with optimum growth temperatures between 23 and 25 degrees C, degraded tetrachlorophenol faster at 8 degrees C than at room temperature indicating distinctly different temperature optima for chlorophenol degradation and growth on complex media. These results show efficient polychlorophenol degradation by the isolates at the boreal groundwater conditions, i.e., at low temperature and low oxygen concentrations. Differences in chlorophenol degradation and sensitivities to chlorophenols and oxygen among the isolates indicate that the phylogenetically different chlorophenol degraders have found different niches in the contaminated groundwater and thus potential for contaminant degradation under a variety of saturated subsurface conditions.

Subtercola boreus gen. nov., sp. nov. and Subtercola frigoramans sp. nov., two new psychrophilic actinobacteria isolated from boreal groundwater.

Psychrophilic actinobacterial isolates from permanently cold groundwater in Finland were characterized using a polyphasic approach. Growth on agar plates was observed at temperatures down to -2 degrees C, with an optimum at 15-17 degrees C, but no growth was observed at 30 degrees C. The peptidoglycan type was B2y and the characteristic diamino acid was diaminobutyric acid. The cell wall sugars of strain K265T were rhamnose, ribose, xylose and mannose and those of strain K300T were glucose, rhamnose and xylose. The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and two glycolipids. The main whole-cell fatty acids were 12-methyltetradecanoic acid, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid. Large amounts of anteiso-1,1-dimethoxy-pentadecane and also iso-1,1-dimethoxyhexadecane were present as diagnostic markers. The predominant menaquinones were MK-9 and MK-10. The G+C content of the DNA of strains K265T and K300T was 64.4 and 67.8 mol%, respectively. Comparison of 16S rRNA gene sequences revealed that strains K265T and K300T represent a new lineage among the type-B-peptidoglycan actinomycetes. The closest relatives were Clavibacter michiganensis, Frigoribacterium faeni and Rathayibacter rathayi. On the basis of 16S rDNA sequence, G+C content and chemotaxonomical and physiological characteristics, K265T and K300T clearly represent a new genus. The genus Subtercola gen. nov. is described, together with two species, namely Subtercola boreus sp. nov. (type strain K300T = DSM 13056T = CCUG 43135T) and Subtercola frigoramans sp. nov (type strain K265T = DSM 13057T = CCUG 43136T).

Diversity of chlorophenol-degrading bacteria isolated from contaminated boreal groundwater.

Chlorophenol-degrading bacteria from a long-term polluted groundwater aquifer were characterized. All isolates degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol at concentrations detected in the contaminated groundwater (< 10 mg 1(-1)). Pentachlorophenol was degraded by three isolates when present alone. In two gram-positive isolates, 2,3,4,6-tetrachlorophenol was required as an inducer for the degradation of pentachlorophenol. The gram-positive isolates were sensitive to pentachlorophenol, with an IC50 value of 5 mg/l. Isolates belonging to the Cytophaga/Flexibacter/Bacteroides phylum had IC50 values of 25 and 63 mg/l. Isolates belonging to alpha-, beta- and gamma-Proteobacteria generally tolerated the highest pentachlorophenol concentrations (> 100 mg/l). Polychlorophenol-degrading capacity was found in strains of Nocardioides, Pseudomonas, Ralstonia, Flavobacterium, and Caulobacter previously not known to degrade polychlorophenols. In addition, six polychlorophenol-degrading sphingomonads were found.