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We describe the first freshwater members of the class Actinobacteria that have been isolated. Nine ultramicro-size (<0.1 m 3 ) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 m 3 and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae. The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp. strain DS.The majority of the bacterial cells present in the bacterioplankton of marine and freshwater habitats are small (cell length, Ͻ1.5 m; volume, Ͻ0.3 m 3 ). Even very small bacteria, which are termed ultramicrobacteria (Ͻ0.1 m 3 ), are always present, and they frequently represent the numerically predominant fraction of typical marine and freshwater bacterioplankton. In contrast, in culture the cells of almost all bacteria isolated from bacterioplankton are much larger (cell length, Ͼ1.5 m; volume, Ͼ0.3 m 3 ) than most planktonic bacteria. In the case of several normally large-cell-size bacterial species, it has been shown that under strong starvation conditions the cell size decreases to dimensions typical of the majority of planktonic bacteria (23,26,27,31). There are only a few bacterial strains that have been isolated from marine and soil habitats whose cells are in the ultramicro size range (volume, Ͻ0.1 m 3 ) during growth under lab conditions (4,6,19,20,29,32,33,38). The most intensively investigated ultramicrobacterium is Sphingopyxis alaskensis (formerly Sphingomonas alsakensis [10,37]), a marine bacterium belonging to the class Alphaproteobacteria. Strains of this species have been isolated from Resurrection Bay, Alaska, from the North Sea, and from coastal waters near Japan (6,32,33). Recently, Rappé et...

The influence of altitude and salinity on bacterioplankton community composition (BCC) in 16 highmountain 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.Saline lakes constitute 45 percent of total inland water (running and stagnant waters) (47). Despite the quantitative importance of saline lakes, only a few studies have investigated the diversity of bacterioplankton in such habitats (13,15,23). By contrast, the influence of salinity on bacterioplankton community composition (BCC) in dynamic saline systems, such as estuaries (6,11,12,25,26,37) and coastal solar salterns (3,7,8), has been well investigated. Therefore, the current knowledge on the influence of salinity on BCC is almost completely based on investigations of systems characterized by rapid changes in salinity. These systems are too dynamic to allow inhabitants to evolutionarily adapt to the changing environment. All studies on such systems indicate that salinity strongly controls BCC, i.e., changes in salinity are assumed to result in replacement of suboptimally adapted taxa by taxa better adapted to the current salinity conditions. By contrast, the slow evolution of large saline lakes from freshwater lakes may have allowed bacterial taxa originally adapted to freshwater conditions to adapt to saline conditions. In order to reveal the influence of salinity on the BCC of stagnant inland waters, 16 lakes located at the Qingha...

Lake Taihu is characterized by its shallowness (mean depth = 1.9 m) and large surface area (2,338 km 2 ). Runoff sources are mostly from the mountainous west and southwest, and outflows are located throughout East Taihu. This causes shorter retention times in the south. In contrast, urban pollutants discharge into northern Taihu and result in poor water quality. Non-point pollution from rural areas and sewage wastewater is the primary pollution source. Water current velocity ranges from 10-30 cm s -1 , and surface currents normally follow wind direction. Bottom currents appear to be a compensation flow. Most wave heights are less than 40 cm, and underwater irradiance correlates to seston in the water column. Lacustrine sediment is distributed in littoral zones, mostly along the western shoreline, with almost no accumulation in the lake center. Intensive aquaculture in East Taihu caused eutrophication and hampered water supply in surrounding areas. In addition, development of marshiness in the eastern littoral zones and East Taihu has occurred. The function of flood discharging of East Taihu has been limited by flourishing macrophytes. The problems facing in Lake Taihu will be alleviated by improving the management of nutrient sources into the lake.

bEver since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes, this method has been regarded as the "gold standard" in both microbial phylogeny and ecology studies. However, intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study, 2,013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species), with 87.5% of the divergence detected being below the 1% level. In particular, some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that, at the unique level, full-length 16S rRNA genes can produce an overestimation of as much as 123.7%, while at the 3% level, an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions, with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods, due to its intragenomic heterogeneity, but also recommends that, for bacteria, this overestimation be minimized using primers targeting the V4 and V5 regions. F or decades, 16S rRNA genes, which encode the small subunit of rRNA in prokaryotes, have been widely used in taxonomic assignment and phylogenetic relationship determination (1, 2). The specific properties of the 16S rRNA gene, including its ubiquitous distribution, mosaic structure (3), and relative stability (4), qualify it as an optimal choice to fulfill these applications. Although some argue that 16S rRNA genes alone may not be sufficient to identify closely related species (5, 6) and the use of monocopy genes like rpoB to perform similar studies has been proposed (7), 16S rRNA genes are undoubtedly the most widely used molecular markers in microbial ecological studies due to well-maintained databases (8) and their easy accessibility.For many years, researchers have been trying to estimate the microbial diversity of complex environments, such as soil (9), marine systems (10), and animal gut systems (11, 12). Various techniques have been developed, from culture-dependent methods to 16S rRNA genes-based methods of clone library (13, 14), denaturing gradient gel electrophoresis (DGGE) (15), terminal restriction fragment length polymorphism (T-RFLP) (16), and the recently developed next-generation sequencing (17). However, the question of how diverse an e...

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Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.Microdiversity of prokaryotes, i.e., the genetic diversity within species-like (Ͼ97% similarity of 16S rRNA genes) phylogenetic groups, receives increasing attention in microbial ecology (1,5,18,19,24,28); however, the ecological significance of this diversity is still unknown. The coexistence of different bacterial genotypes belonging to the same species-like phylogenetic group is well documented for marine (1,18,19,35,45) and freshwater habitats (8,9,10,11,27,30,43). Recently, Acinas et al. (1) demonstrated by the construction and analysis of 16S rRNA clone libraries that a coastal bacterioplankton community contained a very high diversity of ribotypes, the vast majority of which fell into phylogenetically microdiverse sequence clusters (Ͻ1% divergent 16S rRNA sequences). Similarly, extensive microdiversities were also observed in populations of sulfate-reducing bacteria inhabiting a salt marsh (29) and in a Vibrio splendidus population from coastal bacterioplankton (42). The V. splendidus population consisted of at least a thousand distinct genotypes, which demonstrated a high variability in genome size and allelic composition (42).The major aim of the study presented here was the investigation of the intraspecific structure (microdiversity) of a bacterial p...

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