| 1. |
- Fuhrman, J.A., et al.
(författare)
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Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies
- 1988
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 54:6, s. 1426-1429
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Tidskriftsartikel (refereegranskat)abstract
- We developed a simple technique for the high-yield extraction of purified DNA from mixed populations ofnatural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecularbiological approaches to the study of microbial communities in nature. The microorganisms from near-shoremarine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-,um-pore-sizefluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNAwas extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100°C for 1.5 to2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNAwas purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresisshowed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA wassufficiently pure to allow complete digestion by the restriction endonuclease Sau3AI and ligation to vector DNA.In a sample in which the extracted DNA was quantified by binding to the dye Hoechst H33258, DNA wasquantitatively extracted, and 45% of the initially extracted DNA was recovered after purification. Final yieldswere a few micrograms of DNA per liter of seawater and were roughly 25 to 50% of the total bacterial DNAin the sample. Alternatives to the initial harvest by filtration method, including continuous-flow centrifugationand thin-channel or hollow-fiber concentration followed by centrifugation, were less efficient than filtration interms of both time and yield, largely because of the difficulty of centrifuging the very small bacteria typical ofmarine plankton. These methods were judged to be less appropriate for studies of natural populations as theyimpose a strong selection for the larger bacteria.
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| 2. |
- Larsson, Per, et al.
(författare)
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Microbial degradation of xenobiotic, aromatic pollutants in humic water
- 1988
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 54:7, s. 1864-1867
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Tidskriftsartikel (refereegranskat)abstract
- The microbial degradation of a number of 14C-labeled, recalcitrant,aromatic pollutants, including trichloroguaiacol and di-, tri-,and pentachlorophenol, was investigated in aquatic model systemsin the laboratory. Natural, mixed cultures of microorganismsin the water from a brown-water lake with a high content ofhumic compounds mineralized all of the tested substances toa higher degree than did microorganisms in the water from aclear-water lake. Dichlorophenol was the most rapidly degradedpollutant.
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| 3. |
- Norquist, A., et al.
(författare)
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Protection against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.
- 1989
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 55:6, s. 1400-1405
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Tidskriftsartikel (refereegranskat)abstract
- The fish pathogen Vibrio anguillarum causes a lethal infectionin rainbow trout (Salmo gairdneri). Three different avirulentmutants, constructed by transposon insertion mutagenesis (VAN20and VAN70) or as antibiotic-resistant mutants (VAN1000), wereisolated by screening 200 individual isolated mutants for avirulence.When used as live vaccines, all three avirulent mutants wereable to induce protective immunity against the homologous aswell as a heterologous strain of V. anguillarum. When VAN1000was used, protective immunity could be recorded 1 week afterbath vaccination with 10(7) bacteria per ml of water for 30min. A single-dose immunization was effective for at least 12weeks. Western immunoblotting showed that strains of V. anguillarumhave antigenic determinants in common with Aeromonas strains.Therefore, we tested and confirmed that VAN1000 also was ableto induce protective immunity against challenge with Aeromonassalmonicida.
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| 4. |
- Rehnstam, Ann-Sofi, 1959-, et al.
(författare)
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Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe
- 1989
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 55:8, s. 1907-1910
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Tidskriftsartikel (refereegranskat)abstract
- 16S rRNA from seven different Vibrio anguillarum strains was partially sequenced and compared. From this sequence information we could design a 25-base-long oligonucleotide and use it as a specific probe for identification of V. anguillarum. This was determined by RNA-DNA colony hybridization and slot-blot hybridization. Strong, specific hybridization to the probe was observed for all V. anguillarum strains tested. Furthermore, no cross-hybridization could be seen against five other bacterial species. The detection limit was 5 x 10(3) bacteria per ml. It was even possible to detect V. anguillarum, by slot-blot hybridization, directly in a homogenized kidney from a fish that had died of vibriosis. The partial sequence information revealed small but significant differences between strains of the same species. These sequence differences are sufficiently significant to allow serotyping on the RNA level. Comparing strains of different serotypes revealed a 10-base and an 11-base difference in V. anguillarum serotypes O8 and O9, respectively, in a 122-base partial sequence.
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| 5. |
- Rehnstam, A.-S., et al.
(författare)
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Identification of Vibrio anguillarum in fish using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe.
- 1989
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 55:8, s. 1907-1910
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Tidskriftsartikel (refereegranskat)abstract
- 16S rRNA from seven different Vibrio anguillarum strains waspartially sequenced and compared. From this sequence informationwe could design a 25-base-long oligonucleotide and use it asa specific probe for identification of V. anguillarum. Thiswas determined by RNA-DNA colony hybridization and slot-blothybridization. Strong, specific hybridization to the probe wasobserved for all V. anguillarum strains tested. Furthermore,no cross-hybridization could be seen against five other bacterialspecies. The detection limit was 5 x 10(3) bacteria per ml.It was even possible to detect V. anguillarum, by slot-blothybridization, directly in a homogenized kidney from a fishthat had died of vibriosis. The partial sequence informationrevealed small but significant differences between strains ofthe same species. These sequence differences are sufficientlysignificant to allow serotyping on the RNA level. Comparingstrains of different serotypes revealed a 10-base and an 11-basedifference in V. anguillarum serotypes O8 and O9, respectively,in a 122-base partial sequence.
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| 6. |
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| 7. |
- Wikner, Johan, 1961-, et al.
(författare)
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Use of genetically marked minicells as a probe in measurement of predation on bacteria in aquatic environments
- 1986
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 52:1, s. 4-8
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Tidskriftsartikel (refereegranskat)abstract
- Minicells produced by Escherichia coli M2141 were used as probes to measure predation on pelagic bacteria in situ. The minicells, labeled with [35S]methionine in one specific protein, were shown to disappear in the presence of a microflagellate (Ochromonas sp.), as seen by a decrease in the amount of labeled marker protein with time. Incubation in filtered (pore size, 0.2 μm) and autoclaved seawater did not affect the amount of labeled marker protein in the minicell. The generation time of flagellates feeding on minicells was determined to be similar to that found for flagellates grown on seawater bacteria or living E. coli NC3. Data indicate that minicells are seen as true food particles by the flagellates. The minicell probe was used in recapture experiments, in which predation in situ on pelagic bacteria was demonstrated. The rate of bacterial production showed a clear covariation with the rate of predation, both in different sea areas and in depth profiles. The obtained results (11 field experiments) showed that the rate of predation, on average, accounts for the consumption of 62% of the bacteria produced.
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| 8. |
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| 9. |
- Odham, G., et al.
(författare)
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Model system for studies of microbial dynamics at exuding surfaces such as the rhizosphere.
- 1986
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Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 52:1, s. 191-196
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Tidskriftsartikel (refereegranskat)abstract
- An autoclavable all-glass system for studying microbial dynamics at permeable surfaces is described. Standard hydrophobic or hydrophilic membranes (46-mm diameter) of various pore sizes were supported on a glass frit through which nutrient solutions were pumped by a peristaltic pump. The pump provided a precisely controlled flow at speeds of 0.5 to 500 ml of defined or natural cell exudates per h, which passed through the membrane into a receiving vessel. The construction allowed a choice of membranes, which could be modified. The system was tested with a bacterium, isolated from rape plant roots (Brassica napus L.), that was inoculated on a hydrophilic membrane filter and allowed to develop into a biofilm. A defined medium with a composition resembling that of natural rape root exudate was pumped through the membrane at 0.5 ml/h. Scanning electron microscopic examinations indicated that the inoculum formed microcolonies embedded in exopolymers evenly distributed over the membrane surface. The lipid composition and content of poly-beta-hydroxybutyrate in free-living and adhered cells were determined by gas chromatography. The bacterial consumption of amino acids in the exudate was also studied.
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