Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea

Abstract

The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 microM each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-DL-homoserine lactone at a low concentration of 10 microM. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.

FIG. 1.

Vertical profiles of physicochemical and microbiological parameters in the Gotland Deep on 14 July 2000. (A) Temperature, salinity, and density; (B) oxygen and chlorophyll concentrations (in relative fluorescence units); (C) vertical distribution of total cell counts (TCC) and CFU on agar solidified media. Zone I: warm, photic zone; zone II: cold, aphotic zone; zone III: halocline-oxycline; zone IV: anoxic zone with high salinity.

FIG. 2.

Effect of signal compounds (cAMP, OHHL, and BHL), oxygen, and carbon substrates on the cultivation efficiency of bacteria from different depths of the Gotland basin. Cultivation efficiency was determined by the MPN technique and is given as a percentage of total cell counts. (A) Growth in carbon monomer medium, oxic conditions; (B) growth in polymer medium, oxic conditions; (C) growth in monomer medium, anoxic conditions; (D) growth in polymer medium, anoxic conditions. Controls consisted of the corresponding basal medium containing monomers or polymers but no signal compounds. Horizontal bars indicate 95% confidence intervals. Significant increases in cultivation success versus controls are marked by asterisks (*, P < 0.05; **, P < 0.01). Numbers on the right denote different water bodies (see Fig. 1 legend for zone definitions).

FIG. 3.

DGGE analysis of 16S rDNA fragments of the natural community at 200 m in depth and of bacteria cultured in liquid dilution series (ABWP medium, oxic incubation; compare results shown in Fig. 2B) from the same sample. The arrow indicates the band of the natural community which corresponds to band 1, the fingerprint of the isolated strain G100. Band 2 denotes a fingerprint which was obtained in the presence of cAMP but not with the control.

FIG. 4.

Phylogenetic affiliation of strain G100 within the α subclass of Proteobacteria. GenBank accession numbers are shown in parentheses. The bar represents 2% sequence divergence. Bootstrap values are based on 1,000 resamplings.

FIG. 5.

Quantification of genomic DNA of strain G100 in the natural bacterial community of 200 m in depth by dot blot hybridization. Different concentrations of template DNA of strain G100 served as standards. As a control, DNA of strain G100 was mixed with a constant amount (6.3 ng) of template DNA from the natural community. A digitized image of an exposed Lumi-Film is shown. Values below dots indicate the concentration of genomic DNA employed.

FIG. 6.

Resuscitation of strain G100 after starvation at 4°C under oxic conditions, measured as the increase in nephelometric turbidity units (NTU). (A) Regrowth after the addition of 10 μM cAMP (○) or AMP (▴); (B) regrowth after the addition of BHL (▪) or butyric acid plus homoserine lactone (□).