The power of species sorting: local factors drive bacterial community composition over a wide range of spatial scales.

There is a vivid debate on the relative importance of local and regional factors in shaping microbial communities, and on whether microbial organisms show a biogeographic signature in their distribution. Taking a metacommunity approach, spatial factors can become important either through dispersal limitation (compare large spatial scales) or mass effects (in case of strongly connected systems). We here analyze two datasets on bacterial communities [characterized by community fingerprinting through denaturing gradient gel electrophoresis (DGGE)] in meso- to eutrophic shallow lakes to investigate the importance of spatial factors at three contrasting scales. Variation partitioning on datasets of both the bacterial communities of 11 shallow lakes that are part of a strongly interconnected and densely packed pond system <1 km apart, three groups of shallow lakes approximately 100 km apart, as well as these three groups of shallow lakes combined that span a large part of a North-South gradient in Europe (>2,500 km) shows a strong impact of local environmental factors on bacterial community composition, with a marginal impact of spatial distance. Our results indicate that dispersal is not strongly limiting even at large spatial scales, and that mass effects do not have a strong impact on bacterial communities even in physically connected systems. We suggest that the fast population growth rates of bacteria facilitate efficient species sorting along environmental gradients in bacterial communities over a very broad range of dispersal rates.

Effect of salinity on temporal and spatial dynamics of ammonia-oxidising bacteria from intertidal freshwater sediment.

Temporal and spatial dynamics within an ammonia-oxidising community from intertidal, freshwater sediments were studied in microcosms simulating flooding twice a day with fresh, brackish and marine waters. The microcosms had been filled with the upper 5 cm of intertidal freshwater sediment from the river Scheldt. Changes in community composition were examined by denaturing gradient gel electrophoresis of amplified DNA from the community. In the first week of incubation the initially present members of the Nitrosomonas oligotropha lineage were replaced by other members of the same lineage in the top layer of the sediment subjected to flooding with freshwater. Prolonged incubation extended niche differentiation to a depth of 5 cm. In the microcosms flooded with saline media, the initially present members of the N. oligotropha lineage were replaced by strains belonging to the Nitrosomonas marina lineage, but only in the top 1cm. Shift in community composition occurred earlier in the marine microcosms than in the brackish microcosms and was slower than the change in the freshwater microcosms. Irrespective of the nature of the flooding medium, shifts in community composition were always consistent among replicate microcosms. We conclude that salinity is an important steering factor in niche differentiation among ammonia-oxidising bacteria and also that changes within the community of this functional group of bacteria may occur at different rates.

New DGGE strategies for the analyses of methanotrophic microbial communities using different combinations of existing 16S rRNA-based primers.

Methane-oxidising microbial communities are studied intensively because of their importance for global methane cycling. A suite of molecular microbial techniques has been applied to the study of these communities. Denaturing gradient gel electrophoresis (DGGE) is a diversity screening tool combining high sample throughput with phylogenetic information of high resolution. The existing 16S rRNA-based DGGE assays available for methane-oxidising bacteria suffer from low-specificity, low phylogentic information due to the length of the amplified fragments and/or from lack of resolving power. In the present study we developed new combinations of existing primers and applied these on methane-oxidising microbial communities in a freshwater wetland marsh. The designed strategies comprised nested as well as direct amplification of environmental DNA. Successful application of direct amplification using combinations of universal and specific primers circumvents the nested designs currently used. All developed assays resulted in identical community profiles in wetland soil cores with Methylobacter sp. and Methylocystis sp.-related sequences. Changes in the occurrence of Methylobacter-related sequences with depth in the soil profile may be related to the decrease in methane-oxidizing activity.

A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis.

Denaturant gradient gel electrophoresis (DGGE) is a widely used method for mutation analysis and for studies of microbial diversity. Particular combinations of target gene fragments and primers may give rise to erroneous DGGE profiles. We report on a very straightforward means to eliminate the artifactual 'double bands' that can be encountered in several applications.

Submersed macrophytes play a key role in structuring bacterioplankton community composition in the large, shallow, subtropical Taihu Lake, China.

Within-lake horizontal heterogeneity of bacterioplankton community composition (BCC) was investigated in the large and shallow subtropical Taihu Lake (2338 km(2), maximum depth < 3 m). Samples were collected at 17 sites along a trophic gradient ranging from mesotrophic to hypertrophic areas in August and September 2004. These sites cover two alternative stable states of shallow lakes, which are basically characterized by the dominance or the lack of submerged macrophytes. In the case of Taihu Lake, the macrophyte-dominated state is characterized by clear water and immobilized sediment, and the state largely lacking macrophytes is characterized by the dominance of phytoplankton, frequent wind-driven re-suspension of sediments, and a high turbidity. Three different methods, i.e. denaturing gradient gel electrophoresis (DGGE), reverse line blot hybridization (RLB) with probes targeting 17 freshwater bacterial groups, and 16S rRNA gene cloning and sequencing, were used for analysis of BCC. The BCC varied strongly between the two alternative ecological states, but less pronounced between phytoplankton-dominated sites even spanning chlorophyll a gradients from 16.5 (mesotrophic) to 229.8 microg l(-1) (hypertrophic). The 16S rRNA gene library representing the turbid water state contained many sequences closely related to sequences previously obtained from soil or freshwater sediment samples. Furthermore, sequences representing two new lineages of freshwater Actinobacteria were obtained from the investigated samples. Comparative statistical analyses of BCC along the investigated ecological gradients revealed that the dominance of submersed macrophytes was the most influential factor on BCC, responsible for a major part of the observed within-habitat heterogeneity of BCC in Taihu Lake.

Bacterioplankton community composition along a salinity gradient of sixteen high-mountain lakes located on the Tibetan Plateau, China.

The influence of altitude and salinity on bacterioplankton community composition (BCC) in 16 high-mountain lakes located at altitudes of 2,817 to 5,134 m on the Eastern Qinghai-Xizang (Tibetan) Plateau, China, spanning a salinity gradient from 0.02% (freshwater) to 22.3% (hypersaline), was investigated. Three different methods, fluorescent in situ hybridization, denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing, and reverse line blot hybridization (RLB) with probes targeting 17 freshwater bacterial groups, were used for analysis of BCC. Furthermore, the salt tolerances of 47 strains affiliated with groups detected in or isolated from the Tibetan habitats were investigated. Altitude was not found to influence BCC significantly within the investigated range. Several groups of typical freshwater bacteria, e.g., the ACK-M1 cluster and the Polynucleobacter group, were detected in habitats located above 4,400 m. Salinity was found to be the dominating environmental factor controlling BCC in the investigated lakes, resulting in only small overlaps in the BCCs of freshwater and hypersaline lakes. The relative abundances of different classes of Proteobacteria showed a sharp succession along the salinity gradient. Both DGGE and RLB demonstrated that a few freshwater bacterial groups, e.g., GKS98 and LD2, appeared over wide salinity ranges. Six freshwater isolates affiliated with the GKS98 cluster grew in ecophysiological experiments at maximum salinities of 0.3% to 0.7% (oligosaline), while this group was detected in habitats with salinities up to 6.7% (hypersaline). This observation indicated ecologically significant differences in ecophysiological adaptations among members of this narrow phylogenetic group and suggested ecological significance of microdiversity.

Distribution of typical freshwater bacterial groups is associated with pH, temperature, and lake water retention time.

The distribution of 15 typical freshwater bacterial groups in 15 diverse lakes in northern Europe was investigated using reverse line blot hybridization. Statistical evaluation of the data in relation to the characteristics of the lakes showed that pH, temperature, and the theoretical hydrological retention time of the lakes were most strongly related to variations in the distribution of bacterial taxa. This suggests that pH and temperature are steering factors in the selection of taxa and supports the notion that communities in lakes with short water turnover times are influenced by the input of bacterial cells from the drainage areas. Within the beta subdivision of the Proteobacteria (Betaproteobacteria), as well as within the divisions Actinobacteria and Verrucomicrobia, different subgroups were associated differently with environmental variables.

Contrasting microcystin production and cyanobacterial population dynamics in two Planktothrix-dominated freshwater lakes.

Microcystin concentrations in two Dutch lakes with an important Planktothrix component were related to the dynamics of cyanobacterial genotypes and biovolumes. Genotype composition was analysed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rrn operon (rRNA-ITS), and biovolumes were measured by using microscopy. In Lake Tjeukemeer, microcystins were present throughout summer (maximum concentration 30 microg l(-1)) while cyanobacterial diversity was low and very constant. The dominant phototroph was Planktothrix agardhii. In contrast, Lake Klinckenberg showed a high microcystin peak (up to 140 microg l(-1)) of short duration. In this lake, cyanobacterial diversity was higher and very dynamic with apparent genotype successions. Several genotypes derived from DGGE field profiles matched with genotypes from cultures isolated from field samples. The microcystin peak measured in Lake Klinckenberg could be confidently linked to a bloom of Planktothrix rubescens, as microscopic and genotypic analysis showed identity of bloom samples and a toxin-producing P. rubescens culture. Toxin-producing genotypes were detected in the microbial community before they reached densities at which they were detected by using microscopy. Cyanobacterial biovolumes provided additional insights in bloom dynamics. In both lakes, the microcystin content per cell was highest at the onset of the blooms. Our results suggest that while genotypic characterization of a lake can be valuable for detection of toxic organisms, for some lakes a monitoring of algal biomass has sufficient predictive value for an assessment of toxin production.

Molecular characterization of cyanobacterial diversity in a shallow eutrophic lake.

We have studied the diversity of pelagic cyanobacteria in Lake Loosdrecht, The Netherlands, through recovery and analysis of small subunit ribosomal RNA gene sequences from lake samples and cyanobacterial isolates. We used an adapted protocol for specific amplification of cyanobacterial rDNA for denaturing gradient gel electrophoresis (DGGE) analysis. This protocol enabled direct comparison of cyanobacterial community profiles with overall bacterial profiles. The theoretical amplification specificity of the primers was supported by sequence analysis of DNA from excised DGGE bands. Sequences recovered from these bands, in addition to sequences obtained by polymerase chain reaction (PCR) and cloning from lake DNA as well as from cyanobacterial isolates from the lake, revealed a diverse consortium of cyanobacteria, among which are representatives of the genera Aphanizomenon, Planktothrix, Microcystis and Synechococcus. One numerically important and persistent cyanobacterium in the lake, Prochlorothrix hollandica, appeared to co-occur with an unknown but related species. However, the lake is dominated by filamentous species that originally have been termed 'Oscillatoria limnetica-like'. We show that this is a group of several related cyanobacteria, co-occurring in the lake, which belong to the Limnothrix/Pseudanabaena group. The available variation among the coexisting strains of this group can explain the persistent dominance of the group under severe viral pressure.

High-resolution differentiation of Cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis.

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis: Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.

Toxic and nontoxic microcystis colonies in natural populations can be differentiated on the basis of rRNA gene internal transcribed spacer diversity.

Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.

Competition of a parathion-hydrolyzing Flavobacterium with bacteria from ditch water in carbon-, nitrate- and phosphate-limited continuous cultures.

Abstract The effect of competition for macroelements with bacteria from ditch water on the parathion-hydrolyzing Flavobacterium sp. ATCC 27551 (FB) was investigated within mixed continuous cultures under carbon-, nitrate- or phosphate-limited conditions. The high initial rate of parathion hydrolysis decreased rapidly in all cultures due to the loss of strain FB. Addition of 2-isopropyl-6-methyl-4-pyrimidinol (a selective source of carbon, nitrogen and energy for FB) to one nitrate- and carbon-limited chemostat caused a 20-fold increase in parathion-hydrolyzing activity compared to unamended control cultures and retention of FB. The presence of the parathion hydrolase-encoding gene could be demonstrated by a newly developed PCR detection method in all FB cultures during most of the cultivation period. These results suggest that competition effects cause the pesticide-degrading capacity of microbial communities depending on their frequency of exposure to the pesticide compounds.

Rapid screening for freshwater bacterial groups by using reverse line blot hybridization.

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.