A combination of cultivation-based methods with a molecular biological approach was used to investigate whether planktonic bacteria with identical 16S rRNA gene sequences can represent distinct eco-and genotypes. A set of 11 strains of Brevundimonas alba were isolated from a bacterial freshwater community by conventional plating or by using a liquid most-probable-number (MPN) dilution series. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction, as well as in the highest positive dilutions of the MPN series. However, internally transcribed spacer and enterobacterial repetitive intergenic consensus PCR fingerprinting analyses, as well as DNA-DNA hybridization analyses, revealed great genetic diversity among the 11 strains. Each strain utilized a specific combination of 59 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. In dialysis cultures incubated in situ, each strain had a different growth rate and cell yield. We thus demonstrated that the B. alba strains represent distinct populations with genetically determined adaptations and probably occupy different ecological niches. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences.Analysis of 16S rRNA gene sequences has become the primary approach for studying the natural occurrence and distribution of bacteria in a culture-independent manner (3). The vertical and seasonal distributions of distinct 16S rRNA gene sequences (phylotypes) within one ecosystem have been used to infer the ecological niches of bacteria (40,56). This approach is especially valuable if the physiology of bacteria that have not been cultured yet is to be elucidated.In many cases phylogenetically closely related bacteria (whose 16S rRNA sequences differ by between 2.7 and 0.3%) have been detected in the same freshwater, marine, or soil habitat (19,32,43,56). According to macroecological principles of competitive exclusion, physiologically similar microorganisms should not co-occur in nutrient-poor systems which are dominated by physical and chemical fluctuations (40). Accordingly, phylogenetically closely related bacteria coexisting in the same habitat occupy distinct ecological niches (19,32,43,56).For pathogenic bacteria it is well established that even phylogenetically identical strains or species can exhibit distinct ecophysiological properties. Certain serovars of Mycobacterium intracellulare (10), serovars of Ochrobactrum anthropi (27), strains of Yersinia pestis and Yersinia pseudotuberculosis (55), or strains of Bacillus anthracis and Bacillus cereus (4) contain identical 16S rRNA gene sequences. These phylogenetically identical organisms are also genetically highly similar based on DNA-DNA hybridization data (Table 1) but clearly represent different ecotypes based on their virulence properties or host ranges. Often, p...

We have analysed the inter- and intra-lake variability of free-living and particle-associated freshwater Actinobacteria communities in four limnological different lakes of the Mecklenburg Lake District, Northeastern Germany. Denaturing gradient gel electrophoresis (DGGE) specific for Actinobacteria was used to investigate phylogenetic diversity and seasonal dynamics of actinobacterial communities in the epilimnion of all lakes (inter-lake variability) and to assess differences between Actinobacteria communities of the epi-, meta- and hypolimnion of a single lake (intra-lake variability) respectively. DGGE analyses showed significant inter- and intra-lake differences between Actinobacteria communities of all lakes and water layers as well as between free-living and particle-associated Actinobacteria. Phylogenetic inferences of 16S rRNA gene sequences suggest that particular members of particle-associated Actinobacteria were exclusively affiliated to certain actinobacterial lineages. The phylogenetic comparison of 16S rRNA gene sequences of all lakes and water layer, however, indicated the occurrence of almost similar phylogenetic lineages in all studied habitats and suggest high intracluster diversity within already known actinobacterial lineages. Non-metric multidimensional scaling (NMS) ordination analyses and Pearson's product moment correlations revealed several strong correlations between the investigated Actinobacteria communities and various limnological parameters, such as conductivity, total phosphorous, alkalinity or primary production. However, no uniform correlation patterns were found between lakes, water layers and bacterial fractions. These heterogeneous correlation patterns together with the phylogenetic similarities of Actinobacteria communities from different lakes indicate that particular Actinobacteria represent various ecotypes or exhibit a pronounced ecophysiological plasticity.

The diversity of cultured planktonic bacteria was analyzed. Bacterial strains were isolated from a eutrophic lake (Zwischenahner Meer, Niedersachsen, Germany) at three different sampling dates (October 1997, April and May 1998). Phylogenetic diversity was assessed by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and sequencing of 16S rRNA gene fragments. Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed a high genomic diversity within the strain collections, which exceeded the diversity of the 16S rRNA gene sequences considerably. The composition of each of the three strain collections was unique since strains isolated at different dates always exhibited different ERIC-PCR fingerprints. Growth tests with 59 different carbon substrates demonstrated that even strains with identical ERIC-PCR fingerprints, isolated on one sampling date, differed in their physiology. The culturable fraction investigated in the present study constituted a relatively small fraction (

The diversity of cultured planktonic bacteria was analyzed. Bacterial strains were isolated from a eutrophic lake (Zwischenahner Meer, Niedersachsen, Germany) at three different sampling dates (October 1997, April and May 1998). Phylogenetic diversity was assessed by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and sequencing of 16S rRNA gene fragments. Enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed a high genomic diversity within the strain collections, which exceeded the diversity of the 16S rRNA gene sequences considerably. The composition of each of the three strain collections was unique since strains isolated at different dates always exhibited different ERIC-PCR fingerprints. Growth tests with 59 different carbon substrates demonstrated that even strains with identical ERIC-PCR fingerprints, isolated on one sampling date, differed in their physiology. The culturable fraction investigated in the present study constituted a relatively small fraction (9 15%) of the whole bacterioplankton assemblage. Nevertheless, the high physiological diversity in this fraction already indicates that a multitude of different ecological niches must exist in the planktonic environment. The majority of strains isolated in April prior to the decay of the phytoplankton bloom were members of the Cytophaga-Flavobacterium group. One month later, not a single strain of this group could be isolated. When a group-specific PCR-DGGE technique was employed, rapid shifts in the diversity of non-cultured Cytophaga-Flavobacteria also became evident. Based on the rapid shifts in the composition of cultivated as well as some non-cultivated bacteria, the ecological niches in the planktonic habitat must undergo rapid temporal changes. ß

A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm ؊1 , mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.