Complete genome sequence of Polynucleobacter necessarius subsp. asymbioticus type strain (QLW-P1DMWA-1(T)).

Polynucleobacter necessarius subsp. asymbioticus strain QLW-P1DMWA-1(T) is a planktonic freshwater bacterium affiliated with the family Burkholderiaceae (class Betaproteobacteria). This strain is of interest because it represents a subspecies with cosmopolitan and ubiquitous distribution in standing freshwater systems. The 16S-23S ITS genotype represented by the sequenced strain comprised on average more than 10% of bacterioplankton in its home habitat. While all strains of the subspecies P. necessarius asymbioticus are free-living freshwater bacteria, strains belonging to the only other subspecies, P. necessarius subsp. necessarius are obligate endosymbionts of the ciliate Euplotes aediculatus. The two subspecies of P. necessarius are the instances of two closely related subspecies that differ in their lifestyle (free-living vs. obligate endosymbiont), and they are the only members of the genus Polynucleobacter with completely sequenced genomes. Here we describe the features of P. necessarius subsp. asymbioticus, together with the complete genome sequence and annotation. The 2,159,490 bp long chromosome with a total of 2,088 protein-coding and 48 RNA genes is the first completed genome sequence of the genus Polynucleobacter to be published and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2006.

Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria).

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approximately 98.5%, have been reported to occur as obligate endosymbionts of ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S-23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex life cycle. These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and obligately free-living bacteria ever revealed.

Low intraspecific diversity in a polynucleobacter subcluster population numerically dominating bacterioplankton of a freshwater pond.

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.

Ecotypes of planktonic actinobacteria with identical 16S rRNA genes adapted to thermal niches in temperate, subtropical, and tropical freshwater habitats.

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6 degrees C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.

The filtration-acclimatization method for isolation of an important fraction of the not readily cultivable bacteria.

We developed a novel method, the filtration-acclimatization method (FAM), which enables the isolation and cultivation of an important fraction of the bacterial diversity, which is not cultivable by standard methods. The method consists of a filtration step, which removes most of the readily cultivable bacteria able to overgrow slowly growing bacteria, and an acclimatization procedure that provides a slow transition from the low environmental substrate concentrations to the high concentration of standard microbial media. So far, we isolated in total 65 strains from surface freshwater habitats by utilizing FAM. The isolates are affiliated with Actinobacteria, Alpha-, Betaproteobacteria, Bacteroidetes, and Spirochaeta. All isolates are pure cultures and form visible colonies on agar plates with high substrate concentrations. For further analysis, strains sharing more than a 97% 16S rRNA gene sequence similarity were grouped into one taxon. Based on sequence similarities, 88% of the obtained taxa can be considered to be undescribed species (<97% similarity to closest species). The highest similarity value of the taxa to the respective closest related species ranged from 87.7% to 99.8%, and was on average 94.5%. For comparison we isolated, by direct plating of water samples on a rich agar medium, a similar number of taxa. Amongst these taxa the percentage of taxa, which can be considered to be undescribed species, was only half of the percentage found for the taxa isolated by FAM. More importantly, it was amongst the taxa obtained by the standard method no taxon that was closer related to an uncultured bacterium than to an isolate, while 56% of the taxa isolated by FAM were closely related to uncultured bacteria.

Rapid establishment of clonal isolates of freshwater autotrophic picoplankton by single-cell and single-colony sorting.

We describe single-cell and single-colony sorting protocols which allowed for rapid establishment of a diverse culture collection of clonal autotrophic picoplankton (APP) isolates originating from oligotrophic and oligo-mesotrophic subalpine lakes. Overall sort recoveries, expressed as the percentage of sorted microwells exhibiting APP growth, ranged from 5% to 17% depending on the type of APP, but the growth success varied greatly (from 0% to 68%) depending on the origin of the sorted sample. We applied two direct sequencing and two denaturing gradient gel electrophoresis (DGGE) protocols to identify and characterize the genetic purity of 21 of our picocyanobacteria cultures, namely, direct sequencing of the 16S rRNA gene and cpcBA-IGS region, and DGGE analyses involving a 194-bp fragment of the internal transcribed spacer (ITS) and a ca. 500-bp fragment of the phycocyanin (PC) operon (cpcBA-IGS, novel protocol described herein). Of those 21 picocyanobacteria cultures obtained by single-cell/single-colony sorting and subsequently characterized genetically/screened for genetic purity, only one culture was composed of multiple picocyanobacterial strains.

Dispersal and phylogenetic diversity of nonmarine picocyanobacteria, inferred from 16S rRNA gene and cpcBA-intergenic spacer sequence analyses.

More than 20 Synechococcus and Cyanobium isolates were obtained from central European subalpine lakes and sequenced for their 16S rRNA gene and part of the phycocyanin operon (cpc), specifically the intergenic spacer (IGS) between cpcB and cpcA, and corresponding flanking regions (cpcBA-IGS). Maximum-likelihood analyses revealed the existence of at least six to seven clusters of nonmarine picocyanobacteria within the picophytoplankton clade and support the conjecture of global dispersal for some closely related picocyanobacterial genotypes.