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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Actinobacteria of the acI lineage are often the numerically dominant bacterial phylum in surface freshwaters, where they can account for > 50% of total bacteria. Despite their abundance, there are no described isolates. In an effort to obtain enrichment of these ubiquitous freshwater Actinobacteria, diluted freshwater samples from Lake Grosse Fuchskuhle, Germany, were incubated in 96-well culture plates. With this method, a successful enrichment containing high abundances of a member of the lineage acI was established. Phylogenetic classification showed that the acI Actinobacteria of the enrichment belonged to the acI-B2 tribe, which seems to prefer acidic lakes. This enrichment grows to low cell densities and thus the oligotrophic nature of acI-B2 was confirmed.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

In temperate lakes, Actinobacteria are often the numerically dominant phylum, where they can constitute > 50% of the total epilimnetic bacteria (Glöckner et al., 2000; Warnecke et al., 2005). Hierarchical classification has named the most prominent lineages (Warnecke et al., 2004; Allgaier and Grossart, 2006; Holmfeldt et al., 2009), and it has been repeatedly shown that among the four major lineages, the lineage acI is the most abundant in the free-living fraction (Allgaier et al., 2007; Rösel et al., 2012). As the most-studied group, the acI lineage was recently subdivided into 13 tribes and one of them contained a named organism (Newton et al., 2011). (This article also laid the new naming structure for freshwater bacteria as follows: phylum/lineage/clade/tribe. To maintain consistency with the taxonomic designation, we are following the terminology outlined in Newton et al., 2011.)

The only formally named member of acI is ‘Ca. Planktophila limnetica,’ which was named based on an enrichment culture that originally contained < 0.1% acI members (Jezbera et al., 2009). The addition of substrates to the enrichment culture increased the proportion of acI, whereby the most effective substrate L-alanine increased the proportion of acI to 5.6% of total cells. With such a low fraction of cultivated bacteria, obtaining the 16S rRNA sequence of the acI was not possible using conventional polymerase chain reaction (PCR)-based clone libraries. It had to be done with specific primers and in two steps, but eventually yielded a sequence within the acI-A clade (tribe ‘Phila’, formerly acI-A2). The rather low proportion of acI also offered little opportunity to obtain much insight into its physiology or lifestyle. Other attempts to elucidate acI ecophysiology have been based on cultivation independent methods (Buck et al., 2009; Philosof et al., 2009; Ghai et al., 2011a,b; Martinez-Garcia et al., 2011; Salcher et al., 2013; Garcia et al., 2013a).

Single-cell genomics yielded the first nearly complete genome sequence from a member of the acI-B1 tribe (Garcia et al., 2013a). Metabolic reconstruction suggested acI bacteria are facultative aerobes with transporters and enzymes for use of pentoses, polyamines and dipeptides. A gene was found encoding for a putative chitinase, adding to the notion that acI Actinobacteria could play a role in the remineralization of N-acetylglucosamine (NAG) (Beier and Bertilsson, 2011). The presence of actinorhodopsin confirmed the potential of acI to be photoheterotrophic bacteria (Martinez-Garcia et al., 2011; Wurzbacher et al., 2012), as previously speculated (Sharma et al., 2009). The genome was very streamlined, and it is among the smallest genomes of a free-living bacteria together with Pelagibacter ubique, the most abundant marine bacterium which was isolated on sterile sea water (Giovannoni et al., 2005). However, in order to study the ecophysiology of acI in greater detail, an isolate or a highly enriched coculture is required.

Different techniques were previously developed to isolate aquatic bacteria such as the dilution culture (Button et al., 1993) and the filtration acclimatization method (Hahn et al., 2004). In an attempt to isolate acI members, we tried several variations and combinations of these methods. Barriers preventing successful isolation of acI Actinobacteria may be diverse. Hypotheses might include slow growth, low cell densities, the need of unknown and/or unstable growth factors and the dependence on the presence of other bacteria. This dependence could exist in the form of provision of otherwise unavailable substrates or degradation/detoxification of otherwise harmful substances. After several cultivation attempts, we succeeded in obtaining an enrichment of acI Actinobacteria, growing solely on sterile-filtered lake water, and here, we provide a detailed description on the most successful enrichment procedure and some phylogenetic and physiological information on one of our most-studied enrichment cultures.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

In an effort to isolate the ubiquitous uncultivated freshwater Actinobacteria of the acI lineage, diluted freshwater samples from the Northeast basin of Lake Grosse Fuchskuhle were incubated in 96-well liquid culture plates. Because one of the hypothetical barriers to acI cultivation included unknown and/or unstable growth factors, we used triple filtered lake water from the Northeast basin of Lake Grosse Fuchskuhle as media. After screening numerous culture plates for the presence of acI 16S rRNA genes (including experiments amended with different carbon sources), a ‘control’ plate that was not amended with any carbon source yielded two positive wells, designated as FNE-F6 and FNE-F8. Subsequent investigation using PCR and sequencing of the quantitative polymerase chain reaction (qPCR) product from the acI assay confirmed the presence of a strain affiliated with the acI Actinobacteria lineage in both enrichment cultures.

One hundred microlitre from the positive wells were propagated into 20 ml of triple filtered lake water. The enrichments were tested after 4 weeks of inoculation using qPCR assays targeting all bacteria and acI. The qPCR assays revealed that 1% and 9% of the 16S rRNA genes belonged to acI in FNE-F6 and FNE-F8 respectively. Aliquots from both enrichments were propagated and cryopreserved for future study at −80°C using 20% glycerol. At that point, it was clear that both FNE-F6 and FNE-F8 represented enrichments that included acI. We focused our cultivation effort on FNE-F8 which showed a higher initial fraction of acI 16S rRNA genes. The dilution cultivation method was applied to FNE-F8 in an effort to obtain a pure acI culture from a simplified community, but none of the screened wells yielded a detectable pure culture, based on qPCR.

With the second propagation of FNE-F8 in unamended sterile lake water, however, an increase in the percentage of acI 16S rRNA genes (rising from 9% to 30%) was observed. The percentage of acI varied according to the batches of triple filtered lake water, with 30% being the lowest percentage observed. Thereafter, 16S rRNA gene clone libraries were constructed using end-point PCR with the FNE-F8 enrichment to reveal the phylogenetic classification of the most abundant community members. Clone libraries were constructed on transfer 3, transfer 6 and transfer 8 of the FNE-F8 enrichment. Consistently, a high number of the recovered sequences belonged to two groups of ubiquitous freshwater bacteria: (i) Actinobacteria tribe acI-B2 and (ii) Polynucleobacter sp. tribe PnecC. A phylogenetic tree including the acI-B2 and the PnecC sequences recovered is shown in Fig. 1. The flanking community in the enrichments varied with the transfers but sequences belonged mainly to other freshwater Actinobacteria and Proteobacteria (acIII, acTH2, gamII and PnecA). We suspect that the flanking community members might be introduced by the tripled filtered lake water. Even though it is tripled filtered, some ultramicrobacteria may remain in the filtrate.

figure

Figure 1. Phylogenetic tree based on 78 16S rRNA sequences from acI Actinobacteria and Betaproteobacteria found in public databases. The tree was constructed after careful manual alignment of the sequences within ARB (Ludwig et al., 2004). Tree building was performed using the maximum likelihood algorithm PHYML employing the filter ECOLI which considers 1575 positions of the alignment. Accession numbers of reference sequences are given in parentheses. Sequence from FNE-F8 enrichment, acI-B2 and PnecC (from this study) are bold. AcI-B1 is highlighted to show the phylogeny of the only analysed genome.

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The acI-B2 cells were visualized using catalysed reported deposition – fluorescence in situ hybridization (CARD-FISH), and cells were slightly curved rods with an average length of 0.66 μm and a maximum length of 1 μm. The diameter varied between 0.2 and 0.4 μm (Fig. 2). This size is comparable with that found in nature (Glöckner et al., 2000). AcI-B2 was enriched from the Northeast basin of Lake Grosse Fuchskuhle during a year in which pH was 5.6, which is lower than the usual 6.5 ± 0.6 (Allgaier and Grossart, 2006; Garcia et al., 2013b). In fact, a previous study showed that members of the clade B2 indeed prefer acidic conditions (Newton et al., 2007). Members of the acI lineage are already known for being free-living cells in freshwater systems, and in our enrichment, acI-B2 also displays this lifestyle. Here, we want to highlight that free-living does not necessarily mean that the cells are completely independent from any interaction with other bacteria. Particularly, aquatic systems provide the opportunity for ‘free-living bacteria’ to exchange metabolic products with other bacteria through diffusion, and hence, they can be coupled in their metabolism (Church, 2009).

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Figure 2. Epifluorescence microscope image of cells of FNE-F8 enrichment. Culture hybridized with the acI-852 probe and counter stained with 4′,6-diamidino-2-phenylindole (DAPI). The image represents an overlay of two images. The probe positive cells (acI-B2) appear with a green colour. Bar 1 μm.

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The FNE-F8 enrichment was analysed in a variety of tests and propagations, and yielded a number of interesting observations. Recovery of growth of acI-B2 from a glycerol frozen stock was challenging, meaning the density to which acI grew was significantly lower. Therefore, all experiments including the ones shown in the figures were performed using aliquots of the enrichment that was not frozen, but transferred to fresh media as needed. For the future, a better cryopreservation method is needed. Overall, the enrichment was transferred to fresh medium multiple times, and the growth of acI was achieved in different batches of collected lake water. However, growth, percentage and obtainable density of acI-B2 in the enrichment varied when using lake water collected at different sampling dates. For this reason and to render all presented results comparable, a large volume (4 l) of water was collected in September 2012 and prepared for its use as growth medium.

To assess the growth rate of the enrichment, qPCR assays were used. Under the given conditions and on sterile lake water, acI-B2 doubled its population size every 20.7 h, whereby at day 7 after inoculation, the population reached maximum density (Fig. 3). With a growth rate of 0.048 h−1, acI-B2 multiplies more slowly than the typical cultivated bacteria, but similarly to other freshwater bacterioplankton (Šimek et al., 2006) and twice as fast as other cultivated oligotrophic marine bacteria (Rappe et al., 2002). Under the conditions of this batch of water, the relative abundance of acI-B2-16S rRNA genes increased to an average of 78%.

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Figure 3. Growth of FNE-F8 enrichment in Grosse Fuchskuhle lake water media as observed using qPCR. For all bacteria, the detection limit is represented by a dotted line. The acI detection limit is 18 copies per μl. For details on the method used check supplemental. Bars represent standard deviation of five independent replicates.

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As mentioned earlier, our results showed that acI-B2 levels in the enrichments varied with the collection time of lake water (ranging from 6 × 102 to 2 × 103 16S rRNA genes μl−1) and presumably by the quality of the lake water, e.g. during leaf litter input (Hutalle-Schmelzer et al., 2010), algae blooms (Grossart and Simon, 2007) or viral outbreaks (Middelboe and Jørgensen, 2006). Interestingly, this has been also found for SAR11 isolates (i.e. Pelagibacter ubique) (Rappe et al., 2002), suggesting that environmental conditions and natural chemical compounds present in the water may control population dynamics. Therefore, we asked whether nutrient and organic matter concentration in source lake water could affect density of acI in the enrichment. However, no significant differences could be observed in acI-B2 population density (Fig. 4) when the lake water was adjusted to triple the dissolved components concentration (i.e. 3×) or diluted by half (i.e. 0.5×). Interestingly, when the medium was diluted by 10fold, acI-B2 density was significantly lower, but only by fivefold. The acI-B2 population grew to a lower density on higher lake water media concentration [dissolved organic carbon (DOC) 6.99 mM], supporting the hypothesis that acI-B2 is characterized by an oligotrophic lifestyle.

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Figure 4. Density of acI Actinobacteria at day 7 under different concentrations of triple filtered lake water as measured by quantification of their 16S rRNA gene copy numbers. Bars represent standard deviation of three independent replicates. 6× lake water has a DOC of 6.99 mM with a standard deviation < 1%.

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Once the growth curve of the enrichment had been characterized, enrichments amended with 0.5 g l−1 of several carbon sources were analysed in order to test for growth responses of acI-B2. Maximal growth of acI-B2 bacteria on 20 organic substrates (Fig. S1) was recorded for triple filtered lake water from September 2012. For carbohydrates, the addition of pentoses (xylose and ribose) led to a significant increase in acI-B2 density, whereas the addition of glucose did not cause a significant difference compared with the unamended culture. These observations nicely confirm the prediction from genomic data analysis that clade acI-B members can use pentoses as growth substrate (Garcia et al., 2013a). The addition of putrescine to the growth medium stimulated a significant enhancement of acI-B2 growth. Putrescine ubiquitously occurs in all plant cells (Bouchereau et al., 1999), and as a bacterial decomposition compound (Kusano et al., 2008). It has been found at high concentrations in coastal seawaters during a phytoplankton bloom (Nishibori et al., 2001). An analysis of the acI-B1 single amplified genome (SAG) revealed a complete set of genes for putrescine uptake and conversion to succinate via the transamination pathway. In fact, there have been reports showing that acI abundance increases during phytoplankton blooms (Zeder et al., 2009; Salcher et al., 2010). Three other substrates (among the 20 examined) – pyruvate, triethylamine and NAG – strongly stimulated acI-B2 growth. Incorporation of NAG had already been reported in experiments using chitin-amended lake water (Beier and Bertilsson, 2011). Some of the preferred substrates of acI-B2 confirm genomic information and field experiments (like pentoses and putrescine). However, the interpretation of the response to supplementation needs to be made with caution. The question remains whether some complex interplay with PnecC or the other minor members of the enrichment could have played an essential role for promoting acI-B2 growth.

Additional experiments were carried out attempting to replace the lake water with a defined media. Addition of 16 mg l−1 NaHCO3 to the triple filtered lake water inhibited acI-B2 growth by 10fold, although no pH change was observed. A mineral media was designed without NaHCO3 and amended with xylose, ribose and putrescine. In this media, acI-B2 grew but only to half the usual density. This is an important starting point towards identifying factors that aid growth and eventually designing a medium in which the acI can grow to higher densities. Other tests done include the role of light in growth of acI-B2. Incubations in darkness were done, and it was observed that the acI-B2 density after 7 days of growth was not significantly different to those under light conditions tested. Whereas addition of autoclaved lake water increased the density of PnecC in the enrichment by 10fold, it inhibited the growth of the acI-B2 population by 10fold (data not shown). Addition of glycerol (Fig. S1) led to a slight inhibition of acI-B2 growth and thus may explain why cryopreserved cultures in glycerol had a poor growth recovery. Many of these facts are feasible explanations as to why acI members have hardly been obtained in previous enrichment cultures. For example, the filtration acclimatization method previously used to obtain ‘Ca. Planktophila limnetica’ contained 16 mg l−1 of NaHCO3 (Hahn et al., 2004; Jezbera et al., 2009) which potentially inhibited acI growth. There is another example where the use of NaHCO3 in incubations might have influenced the conclusions on freshwater Actinobacteria. In the same study, it was reported that freshwater Actinobacteria did not respond to enhanced availability of dissolved organic carbon, but it is most likely that it was inhibited by the high concentrations of NaHCO3 used in the medium (Burkert et al., 2003).

The success of our enrichment approach with members of the acI lineage may be attributed to several factors, including the use of filtered lake water as medium. Screening using qPCR assays as part of the strategy allows high throughput and low detection thresholds. It remains unknown and untested if the presence of other partners in the enrichment represents interactions with implications for their growth dynamics. In our lab, this enrichment method was already proven to repeatedly yield acI positives mainly when using samples (and water) from eutrophic lakes (such as the Northeast basin of Grosse Fuchskuhle). Access to an enrichment that includes the acI lineage in high abundance will provide the opportunity for further genome sequence analysis. Obtaining a pure culture to fully study and understand the ecophysiology and metabolism of ubiquitous and abundant organisms in nature such as acI is highly desired, and our enrichment offers a first step to achieve it.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

We would like to acknowledge JSMC and the German Science foundation (DFG GR 1540/17-1) for funding. KDM would like to thank the US National Science Foundation (CBET 0738039 and MCB-0702395) and the National Institute of Food and Agriculture, United States Department of Agriculture (ID number WIS01516). This material is based upon NTL LTER work supported by the National Science Foundation under Cooperative Agreement #0822700. We thank Philipp Baur for his great help during sampling and Sandra Barchmann for her technical support.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information
FilenameFormatSizeDescription
emi412104-sup-0001-si.docx35K

Fig. S1. Density of acI-16S rRNA gene copies at day 7. The effects of the addition of 0.5 g l−1 of several carbon compounds can be observed. (LW = lake water; negative control = non-inoculated triple filtered lake water; NAG = N-acetylglucosamine).

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