Genotyping of heterotrophic bacteria from the central baltic sea by use of low-molecular-weight RNA profiles

103

Community structure of bacterioplankton was studied during the major growth season for phytoplankton (April to October) in the epilimnion of a temperate eutrophic lake (Lake Plußsee, northern Germany) by using comparative 5S rRNA analysis. Estimates of the relative abundances of single taxonomic groups were made on the basis of the amounts of single 5S rRNA bands obtained after high-resolution electrophoresis of RNA directly from the bacterioplankton. Full-sequence analysis of single environmental 5S rRNAs enabled the identification of single taxonomic groups of bacteria. Comparison of partial 5S rRNA sequences allowed the detection of changes of single taxa over time. Overall, the whole bacterioplankton community showed two to eight abundant (>4% of the total 5S rRNA) taxa. A distinctive seasonal succession was observed in the taxonomic structure of this pelagic community. A rather-stable community structure, with seven to eight different taxonomic units, was observed beginning in April during the spring phytoplankton bloom. A strong reduction in this diversity occurred at the beginning of the clear-water phase (early May), when only two to four abundant taxa were observed, with one taxon dominating (up to 72% of the total 5S rRNA). The community structure during summer stagnation (June and July) was characterized by frequent changes of different dominating taxa. During late summer, a dinoflagellate bloom (Ceratium hirudinella) occurred, with Comamonas acidovorans (β-subclass of the classProteobacteria) becoming the dominant bacterial species (average abundance of 43% of the total 5S rRNA). Finally, the seasonal dynamics of the community structure of bacterioplankton were compared with the abundances of other major groups of the aquatic food web, such as phyto- and zooplankton, revealing that strong grazing pressure by zooplankton can reduce microbial diversity substantially in pelagic environments.

Section: Methodsmentioning

confidence: 99%

Seasonal Dynamics of Bacterioplankton Community Structure in a Eutrophic Lake as Determined by 5S rRNA Analysis

Höfle

Haas

Dominik

1999

103

“…Identification of the other two isolates was done by LMW-RNA profiling (14). LMW-RNA profiles of the two identified isolates, determined with long-range gels used according to the method of Höfle and Brettar (16), showed no differences from the profiles of the type strains of the relevant species. Approximately 700 additional isolates from Lake Plußsee and the mesocosm experiments were obtained in 1989 and 1990 by the described isolation procedure.…”

Section: Methodsmentioning

confidence: 99%

“…As determined by molecular identification methods, the three isolates represented very different taxonomic groups, namely, (i) the phylum containing flavobacteria, bacteroides, and cytophaga for Cytophaga johnsonae PX62; (ii) the ␤-subclass of the Proteobacteria for Comamonas acidovorans PX54; and (iii) the ␥-subclass of the Proteobacteria for Aeromonas hydrophila PU7718. In addition to the three isolates used for the generation of the MAbs, a large set of isolates from Lake Plußsee and the mesocosms was obtained and screened for closely related genotypes by low-molecular-weight (LMW)-RNA pro-filing (14,16). The analysis of a combination of reference strains and closely related isolates from the same environment enabled us to establish the specificities of the generated MAbs and to assess the abundance and distribution of the isolates in their habitat.…”

mentioning

confidence: 99%

Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems

Faude

Höfle

1997

Strain-specific monoclonal antibodies (MAbs) were developed for three different bacterial isolates obtained from a freshwater environment (Lake Plußsee) in the spring of 1990. The three isolates, which were identified by molecular methods, were as follows: Cytophaga johnsonae PX62, Comamonas acidovorans PX54, and Aeromonas hydrophila PU7718. These strains represented three species that were detected in high abundance during a set of mesocosm experiments in Lake Plußsee by the direct analysis of low-molecular-weight RNAs from bacterioplankton. We developed one MAb each for the bacterial isolates PX54 and PU7718 that did not show any cross-reactivity with other bacterial strains by immunofluorescence microscopy. Each MAb recognized the general lipopolysaccharide fraction of the homologous strain. These MAbs were tested successfully for their ability to be used for the in situ detection and counting of bacteria in lake water by immunofluorescence microscopy. During the spring of 1993, A. hydrophila PU7718 showed a depth distribution in Lake Plußsee with a pronounced maximum abundance at 6 m, whereas Comamonas acidovorans PX54 showed a depth distribution with a maximum abundance at the surface. The application of these MAbs to the freshwater samples enabled us to determine the cell morphologies and microhabitats of these strains within their natural environment. The presence of as many as 8,000 cells of these strains per ml in their original habitats 3 years after their initial isolation demonstrated the persistence of individual strains of heterotrophic bacteria over long time spans in pelagic habitats.

“…Whether ammonium or a nitrogenous gas is the dominant end product of nitrate reduction in S. putrefaciens has been an issue of controversy. Nitrate reduction to N 2 O has been reported for S. putrefaciens isolated from the water column of the Baltic Sea, where this group represented 77% of the total number of denitrifying bacteria isolated (3,15). In earlier studies, a strain of S. putrefaciens was designated a dissimilatory ammonium producer capable of reducing nitrate to ammonium under some conditions (35)(36)(37).…”

mentioning

confidence: 97%

Physiology and enzymology involved in denitrification by Shewanella putrefaciens

Krause

Nealson

1997

Nitrate reduction to N 2 O was investigated in batch cultures of Shewanella putrefaciens MR-1, MR-4, and MR-7. All three strains reduced nitrate to nitrite to N 2 O, and this reduction was coupled to growth, whereas ammonium accumulation was very low (0 to 1 mol liter ؊1). All S. putrefaciens isolates were also capable of reducing nitrate aerobically; under anaerobic conditions, nitrite levels were three-to sixfold higher than those found under oxic conditions. Nitrate reductase activities (31 to 60 mol of nitrite min ؊1 mg of protein ؊1) detected in intact cells of S. putrefaciens were equal to or higher than those seen in Escherichia coli LE 392. K m values for nitrate reduction ranged from 12 mM for MR-1 to 1.3 mM for MR-4 with benzyl viologen as an artifical electron donor. Nitrate and nitrite reductase activities in cell-free preparations were demonstrated in native gels by using reduced benzyl viologen. Detergent treatment of crude and membrane extracts suggested that the nitrate reductases of MR-1 and MR-4 are membrane bound. When the nitrate reductase in MR-1 was partially purified, three subunits (90, 70, and 55 kDa) were detected in denaturing gels. The nitrite reductase of MR-1 is also membrane bound and appeared as a 60-kDa band in sodium dodecyl sulfate-polyacrylamide gels after partial purification.