Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton

Abstract

The effect of signal compounds and of different incubation conditions on the culturability (i.e., the fraction of all cells capable of growth) of natural bacterioplankton from the eutrophic lake Zwischenahner Meer was investigated over a period of 20 months. Numbers of growing cells were determined by the most-probable-number technique in liquid media containing low concentrations (10 micro M) of the signal compounds N-(oxohexanoyl)-DL-homoserine lactone, N-(butyryl)-DL-homoserine lactone, cyclic AMP (cAMP), or ATP. cAMP was the most effective signal compound, leading to significantly increased cultivation efficiencies of up to 10% of the total bacterial counts. Microautoradiography with [2,8-(3)H]cAMP, combined with fluorescence in situ hybridization, demonstrated that cAMP was taken up by 18% of all cells. The bacterial cAMP uptake systems had a very low K(m) value of </=1 nM. Analysis of the cultured bacteria by 16S rRNA gene fingerprinting showed that different bacterial phylotypes were recovered in the presence and in the absence of cAMP. Consequently, the addition of cAMP caused a stimulation of otherwise nonculturable bacteria. Phylogenetically different bacteria were also recovered at different temperatures and oxygen partial pressures. Throughout the study period, mainly members of the beta-subclass of the Proteobacteria were cultivated. In addition, some members of the Actinomycetales were enriched. Quantification by culture-independent fluorescence in situ hybridization demonstrated that beta-Proteobacteria and Actinomycetales also dominated the natural bacterioplankton assemblage. Sequence comparison revealed that two members of the Actinomycetales which reached high numbers in the natural bacterioplankton assemblage could actually be enriched by our cultivation approach.

FIG. 1.

Effect of signal compounds on the efficiency of cultivation of planktonic bacteria from Zwischenahner Meer (sampled on 21 October 1999). Cultivation efficiency was determined by the MPN technique and is given as the percentage of total cell counts. Error bars indicate 95% confidence intervals. A significant increase in cultivation success compared to the control (P < 0.01) is indicated by an asterisk. Control compounds were AMP, butyric acid (BA), hexanoic acid (HA), and HSL.

FIG. 2.

Analysis of 16S rRNA gene fingerprints from the highest positive dilutions of MPN series. As an example, results obtained with a sample from 24 April 2001 are shown. On this date, cAMP did not affect the cultivation success. (A) Variability of fingerprints recovered in four different parallels of media containing cAMP or AMP. Arrows indicate fingerprints recovered from media containing cAMP as well as AMP. Asterisks indicate unique fingerprints. (B) Effect of incubation temperature (left panel) and oxygen partial pressure (right panel) on the phylotypes obtained in the highest positive dilutions of media supplemented with either cAMP or AMP.

FIG. 3.

Comparison of 16S rRNA gene fingerprints of bacteria growing in the presence of cAMP or ATP with those detected in control assay mixtures supplemented with AMP. Only MPN series in which bacterial growth was significantly stimulated by cAMP or ATP are shown (compare Table 1).

FIG. 4.

(A) Composition of bacterioplankton communities in Zwischenahner Meer as analyzed by 16S rRNA gene fingerprinting. (B) Total cell numbers (•), water temperature (- - -), and conductivity (. . .) at the same sampling dates as in panel A. (C) Schematic representation of cumulative 16S rRNA gene fingerprints obtained from separate enrichments on different dates, either in the presence of cAMP alone (solid lines) or with AMP or both cAMP and AMP (dotted lines), at all different incubation temperatures and oxygen partial pressures. Dotted arrows on the left of panel A indicate positions at which fingerprints of cultured strains correspond to those of the natural community but, when sequenced, nevertheless contained different sequences. The sequence obtained from band Z34 was identical to that from Z35, and the sequence from band Z8 was identical to that from Z39 (compare Fig. 5).

FIG.5.

Phylogenetic tree calculated by maximum likelihood, comparing 16S rRNA gene sequences recovered from different MPN enrichments and from the natural community of bacteria from Zwischenahner Meer with those of their closest relatives. Sequences obtained in the present study are shaded. cAMP and ATP indicate sequences originating from highest positive dilutions of series in which cAMP or ATP resulted in significantly increased MPN values. Nat. com., natural bacterioplankton community from Zwischenahner Meer. Three of the freshwater clusters (Polynucleobacter necessarius, Rhodoferax sp. strain BAL47, and ACK-M1) suggested by Zwart et al. (52) are identified by brackets. The bar indicates 10% sequence divergence.

FIG. 6.

Uptake of [³H]cAMP by natural freshwater bacterioplankton in water samples from 22 April 2002. (A) Time course of [³H]cAMP uptake. (B) Inhibition of [³H]cAMP uptake by different concentrations of unlabeled cAMP. The curve fit was calculated with a K_m for cAMP uptake of 1 nM, as described in the text.