TY - JOUR
AU - Overmann,, Jörg
AB - Abstract Using the MicroDrop® microdispenser system, a novel approach for high throughput cultivation assays for the determination of numbers of culturable bacteria, and for the isolation of bacteria in liquid media was established. The MicroDrop device works similar to an ink jet printer. Droplets of 150–200 pl are created at the nozzle of a glass micropipette by means of a computer-driven piezo transducer, and are dispensed automatically at predetermined positions with the aid of a XYZ-positioning system. The actual drop volume is highly reproducible and is determined by the pulse duration, the pulse frequency and the micropipette geometry. Culture media in 96-well microtiter plates were inoculated with constant numbers of bacteria from three different natural freshwater lakes. The number of culturable bacteria in the sample can be calculated from the frequency of wells showing bacterial growth, based on a binomial distribution of culturable cells. Our method was compared to the conventional most probable number (MPN) approach, the technique presently most often used for the determination of bacterial culturability and for the isolation of numerically dominant culturable bacteria. As opposed to the MPN technique, our approach yields data with much higher statistical significance (i.e. a 10 times lower standard deviation) due to the higher number of parallels which can be performed in each microtiter plate. The values of culturable bacteria as determined by the MPN and MicroDrop techniques were only weakly correlated (r2=0.570, n=42, P<0.001). Cultivation efficiencies obtained with the MicroDrop technique were systematically lower than MPN values by a factor of 2.7, indicating a significant overestimation of culturability by the latter method. The composition of the cultured bacterial fraction was determined by denaturing gradient gel electrophoresis fingerprinting of 16S rDNA fragments and sequencing. This demonstrated that phylogenetically similar bacteria were recovered by both cultivation techniques. Both methods resulted in the recovery of many previously unknown aquatic bacteria affiliated to the same taxonomic groups and, in one case, in the isolation of a numerically dominant, but not-yet-cultured β-Proteobacterium which was ubiquitous in all three lakes. Bacterioplankton, Cultivation, Culturability, Most probable number technique 1 Introduction One of the most pressing problems in microbial ecology is the small fraction of the total bacterial cells which can be cultivated [1,2]. Generally, less than 1%, and sometimes even as little as 0.001%, of all prokaryotic cells multiply in laboratory media [1]. Since culture-independent studies indicate that the vast majority of the numerically important bacteria have not yet been cultured [3,4], novel cultivation approaches are highly desirable to make progress in our understanding of the structure and function of microbial communities. The fraction of cells capable of multiplication (the so-called ‘culturability’) in a given medium can be quantified as colony forming units on solid media or on filters floating in a liquid medium [5]. Alternatively, culturability can be determined by the most probable number (MPN) method in liquid dilution series [6–11]. In the majority of cases, the MPN method yields higher numbers of culturable cells than plating on solid media [12], although exemptions exist [13]. This may be attributed to the fact that some bacteria do not form colonies at the air–solid interface [2]. So far, MPN series have mostly been employed with highly selective media, e.g. to determine the culturability of sulfate- or nitrate-reducing, methanogenic or sulfur-oxidizing bacteria [7–9], or to isolate thermophilic cyanobacteria [14]. In liquid cultures, dominant bacteria are frequently overgrown by accompanying species which are less abundant, but grow faster. The latter species can easily be eliminated by liquid dilution series. Therefore, the highest positive dilutions of MPN series provide enrichments or even pure cultures of abundant but fastidious bacteria which are missed in conventional enrichment trials [14]. Hence, MPN series also represent an important tool for the isolation of novel prokaryotes. More recently, the suitability of the MPN technique as a tool for the isolation of numerically dominant prokaryotes has been evaluated more systematically [15–18]. In almost all cases, however, the isolates obtained did not correspond to abundant phylotypes in the original microbial community [15,16]. A principal limitation of the MPN method is that only a small number of parallel dilution series (usually 3–10) can be processed for each sample. Consequently, the number of strains which can be potentially isolated from the highest dilutions is in the order of 10 at most. The small number of parallels also causes the inherently large statistical uncertainty of the cultivation efficiencies [15,19]. Because of the above shortcomings, the conventional MPN approach does not seem to be appropriate if the cultivated fraction is considerably diverse and different types of bacteria occur at low frequencies. Instead, the generation of diluted inocula by automated methods and a subsequent screening of large numbers of isolates by molecular fingerprinting methods appears to constitute a more promising approach. Recently, a high throughput technique for extinction culturing was applied to bacterioplankton samples from coastal seawater [20] and yielded many novel microbial strains that may be abundant in the natural habitat. In this method, the inoculum is diluted to a cell titer of 1.1–5 cells ml−1 and 1-ml aliquots are dispensed manually into 48-well microtiter plates. In the present investigation, we established a novel technique for the fully automated inoculation of microtiter plates with defined numbers of microbial cells. By reproducibly generating aliquots of natural samples as small as 170 pl, the MicroDrop technique permits inoculation of 96 samples in less than 1 min, at the same time avoiding an initial dilution of the samples. Growth experiments were performed in five non-selective liquid media supplemented with different signal compounds and the results of our novel approach were compared to those obtained in parallel by the conventional MPN technique. 2 Materials and methods 2.1 Sampling site and sample processing Water samples were obtained from three German lakes of different trophic states. Lake Walchensee (near Garmisch-Partenkirchen, Southern Bavaria) is an oligotrophic alpine lake located 802 m above mean sea level and is characterized by an extraordinary relative depth (maximum depth 198 m, surface area of only 1630 ha). Sampling was conducted from a boat at a site (47°35′N, 11°19′E) located 30 m from the west shore. The mesotrophic prealpine Starnberger See (near Munich, Southern Bavaria) is located 584 m above mean sea level, has a maximum depth of 128 m and a surface area of 5636 ha. Samples were obtained from the head of a 20-m-long pier located on the east shore near the town of Ammerland (47°55′N, 11°02′E). The eutrophic Zwischenahner Meer is a small (surface area of 520 ha), shallow (maximum depth of 5 m, mean depth 3.3 m) lake located in northern Germany near Oldenburg (Niedersachsen) at an altitude of 6 m above mean sea level. The sampling site (53°12′N, 8°0′E) at this lake was located at the head of a pier 20 m from the east shore. On July 17, October 25 2001 and April 29 2002, water samples were obtained from Walchensee and Starnberger See from a depth of 3 m using a bilge pump connected to isoversinic tubing. The inlet of the tubing consisted of two polyvinyl chloride cones spaced 1 cm apart as described previously [21]. Samples from Zwischenahner Meer were collected on July 15, October 14 2001 and April 28 2002 directly at the lake surface since the entire water column of this shallow lake mixes frequently even during summer [18]. All water samples were prefiltered through a 20-μm mesh plankton net. Subsequently, samples were kept at 4°C and processed within 10 h after sampling. Subsamples were fixed in 2% (v/v) glutaraldehyde. 2.2 Total bacterial numbers Fixed water samples were filtered onto 0.1-μm pore size polycarbonate filters (Nucleopore Track-Etch Membrane, Whatman, Springfield Mill, UK). Total bacterial cell counts were determined by epifluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) [22] as described earlier [15]. 2.3 Cultivation by the MPN technique Synthetic freshwater [23] supplemented with a fatty acid mixture containing formate, acetate and propionate (200 μM each, all Na+-salts), an amino acid mixture containing all 20 amino acids (200 μM each), Tween 80 (0.001% v/v), and glucose, pyruvate, citrate, succinate and 2-oxoglutarate (200 μM each, Na+-salts) served as the basal medium. A total of five different media were tested. Four media contained different signal compounds. Either cyclic adenosine monophosphate (Sigma Chemical Co., St. Louis, MO, USA), N-(ketocaproyl)-dl-homoserine lactone (synonymous with N-(oxohexanoyl)-dl-homoserine lactone) (Sigma Chemical Co., St. Louis, MO, USA), N-(butyryl)-dl-homoserine lactone (Fluka Chemie AG, Buchs, Switzerland) or adenosine triphosphate (Sigma Chemical Co., St. Louis, MO, USA) were added to the basal medium to a final concentration of 10 μM. The fifth type of medium did not receive any signal compound and served as control. Aliquots of 180 μl of growth medium were dispensed into sterile 96-well polystyrene microtiter plates (Corning Incorporated, New York, USA). Twenty-μl subsamples of the prefiltered lake water were inoculated into seven wells of each microtiter plate. MPN series were then generated in seven parallels by 1:10 dilutions in consecutive microtiter wells. Per sampling date, one microtiter plate was inoculated for each medium type. After 6 weeks of incubation at 15°C, growth was monitored visually by turbidity and the most probable cell numbers were calculated using a computer program [24]. 2.4 Cultivation approach applying the MicroDrop® technique As an alternative method for the determination of bacterial culturability, we established an automated method for extinction culturing of suspended microbial cells. We employed the MicroDrop® AutoDrop microdispenser system version 5.50 (MicroDrop GmbH, Norderstedt, Germany) (Fig. 1) with which very small droplets can be generated in a reproducible manner. The device works similar to an ink jet printer. Its core consists of a glass capillary (volume, 25 μl) which is surrounded by a piezo actuator. A voltage pulse causes the piezo actuator to contract, thereby creating a pressure pulse in the liquid inside the capillary. At the nozzle of the glass capillary, the pressure wave is transformed into a highly accelerated motion which leads to the expulsion of a small droplet. The droplet diameter was 55–75 μm depending on voltage (100–150 V), pulse duration (26–32 μs) and drop frequency (150 Hz), and the geometry of the micropipette. Droplet generation takes place with a high precision, therefore volume variations are usually less than 1%. In addition, the capillary is connected to a gas pump. By decreasing or increasing the pressure on the liquid within the capillary, it can be filled with fluid or drained. The droplets generated are dispensed automatically at predetermined positions with the aid of a XYZ-positioning system (Fig. 1). Automated dispensing permits an inoculation of 96 samples in less than 1 min. 1 Open in new tabDownload slide Schematic illustration of the MicroDrop® device for automated inoculation of 170-pl aliquots of a bacterial suspension into a 96-well microtiter plate. 1 Open in new tabDownload slide Schematic illustration of the MicroDrop® device for automated inoculation of 170-pl aliquots of a bacterial suspension into a 96-well microtiter plate. The diameters of the droplets were determined under stroboscopic illumination by a video camera integrated in the MicroDrop® system. From the total number of bacterial cells and the calculated volume of single droplets, the number of cells per droplet was calculated assuming that the cells were distributed homogeneously in the water sample. Prior to inoculation of the microtiter plates, the glass capillary was sterilized by repeated flushing with 70% ethanol, then sterile distilled water, and finally was flushed twice with the lake water sample. Based on previous determination of average culturability of the bacterioplankton in the different lakes (1%; [1,17]), each well was inoculated with the same number of droplets such that each individual well received a total of 50 cells. On each microtiter plate, 12 wells were left without inoculation and served as a contamination control. However, contamination of these control wells was never observed. For each of the five different types of growth media (see above), a total of five microtiter plates were inoculated. This resulted in 420 individual growth tests (wells) for each water sample and growth medium. After incubation, the number of wells showing microbial growth was scored and the fraction p of positive wells calculated (wells positive for growth/total inoculated wells). The number of culturable cells per well x and the corresponding 95% confidence interval CI95% were calculated from p and the total number of inoculated wells n, using formulas based on a binomial distribution [25]: 1 2 2.5 PCR amplification and denaturing gradient gel electrophoresis (DGGE) fingerprinting For each lake, between 80 and 120 cultures from the highest positive dilutions of the MPN series or from different wells of the MicroDrop microtiter plates were analyzed by molecular methods. Chromosomal DNA from the cultured bacteria was extracted by the freeze and thaw technique as described previously [15]. 620-bp-long 16S rDNA fragments were amplified using primers GC341f and 907r [26]. Each PCR reaction contained as template 1 μl of the freeze and thaw DNA preparations, 0.5 μM of primers, 200 μM of each deoxyribonucleotide triphosphate, 52 mM KCl, 10.4 mM Tris–HCl, 11.1 mM MgCl2, 0.01% gelatin, and 0.02 U per μl RED Taq DNA polymerase (Sigma Chemical Co., St. Louis, MO, USA). Amplification was performed by step down PCR [27], which includes 10 cycles at an annealing temperature of 62°C, followed by 20 cycles at 57°C. Each cycle started with a melting step at 96°C and ended with an extension step at 72°C. PCR fingerprints were separated by DGGE in 6% (w/v) polyacrylamide gels containing a linear gradient of 30–70% denaturant as described earlier [27]. After staining the gels with ethidium bromide, DNA bands of interest were excised with a sterile scalpel, and the DNA eluted overnight at 4°C in 20 μl of sterile double-distilled water. One μl of the eluate was reamplified under the above mentioned conditions using primer 341f without GC-clamp. The PCR products were purified and quantified for sequencing. The cumulative numbers of melting types (i.e. position of DGGE bands) were used to estimate which fraction of the total diversity of culturable bacterioplankton cells was covered by our 80–120 analyzed samples. The cumulative distribution was plotted using the custom software program RarFac [28]. 2.6 16S rDNA sequencing and phylogenetic analysis Reamplified and purified fragments from DGGE bands were sequenced with the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Perkin Elmer Applied Biosystems GmbH, Weiterstadt, Germany) and the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. The sequencing error of our method was determined to be ≤1 base per 600 nucleotides. The 16S rDNA sequence data were analyzed using the ARB software package (http://www.mikro.biologie.tu-muenchen.de). Sequences of closest relatives were retrieved from the GenBank database employing BLASTN 2.0.4. [29] and imported to the ARB database. The tool Fast Aligner V1.03 was used for automatic sequence alignment which was subsequently checked and corrected manually based on secondary structure information. A phylogenetic tree was constructed by maximum likelihood analyses using only sequences longer than 1200 bp. Subsequently, the shorter sequences obtained from DGGE bands were added by applying the Parsimony tool. Sequence similarities were calculated using the ARB distance matrix. The sequences obtained during the present study have been submitted to the GenBank database under accession numbers AF530916–AF530978, AF530980–AF530989, and AF530991–AF531028. 3 Results and discussion 3.1 Cultivation of natural bacterioplankton applying the MicroDrop and MPN technique The MPN technique is one of the most important current approaches to determine numbers of culturable bacteria and to obtain bacterial isolates from natural sample material [7–10,14,19,30,31]. Routinely, 10-fold dilutions in 3–10 parallels are used, yielding values with an inherently large statistical uncertainty. Similar to previous experience, the MPN data obtained in the present study with single microtiter plates had large 95% confidence intervals. On average, the upper confidence limit reached 500% of the respective MPN value (compare Fig. 2). In contrast, the cultivation efficiencies obtained by the MicroDrop technique with the same number (84) of wells exhibited a 10 times lower uncertainty (upper 95% confidence limit on average ±56% of the mean value). Even if those of the five individual microtiter plates containing the same medium were chosen which yielded the highest statistical uncertainty for MicroDrop data, the upper 95% confidence limits were still eight times smaller than the confidence intervals of the respective MPN values (data not shown). Evidently, the MicroDrop technique is superior to the MPN dilution approach, if the same number of cultures are inoculated. Two-fold instead of 10-fold dilutions may yield somewhat more precise MPN counts [32], but make the inoculation of MPN series even more laborious. Its greater statistical reliability together with its speed and simplicity renders the MicroDrop technique more suitable for testing and improving cultivation conditions for not-yet-cultured bacteria, since increases in cultivation efficiencies can be detected with much higher reliability. 2 Open in new tabDownload slide Comparison of cultivation efficiencies (given as percentage of total bacterial counts) in the presence of cAMP as determined by the MicroDrop () and MPN (◻) techniques. Error bars indicate 95% confidence intervals. Values which were significantly increased over controls without cAMP are marked with asterisks (*P<0.05; **P<0.02; ***P<0.001). 2 Open in new tabDownload slide Comparison of cultivation efficiencies (given as percentage of total bacterial counts) in the presence of cAMP as determined by the MicroDrop () and MPN (◻) techniques. Error bars indicate 95% confidence intervals. Values which were significantly increased over controls without cAMP are marked with asterisks (*P<0.05; **P<0.02; ***P<0.001). When comparing MPN and MicroDrop data obtained with identical media and the same inoculum, the cultivation efficiency attained by the MPN technique significantly surpassed that determined by the MicroDrop technique in more than 50% of the experiments. On average, MPN data were higher by a factor of 15 in these cases. Only in four of all 42 experiments were the MicroDrop data significantly higher than corresponding MPN values, in these cases however only by a factor of 4.5. No statistically significant differences were observed in 16 of the experiments. A correlation analysis of all data (Fig. 3) indicated that, overall, the MicroDrop technique tends to yield cultivation efficiencies which are lower by a factor of 2.7 as compared to the MPN technique (r2=0.570, level of significance P<0.001, Fig. 3). Since significant differences between both techniques occurred mostly in those cases where the MicroDrop counts were lower than the MPN counts (filled circles in Fig. 3), the results obtained by both techniques may differ even by a higher factor than 2.7. 3 Open in new tabDownload slide Correlation analysis of cultivation efficiencies obtained by the MicroDrop and MPN techniques based on the full dataset. Filled circles (●) give pairs of data where MicroDrop values were significantly lower than MPN values. All other data pairs are given as open circles (○). Line indicates linear regression and dotted lines the 95% confidence interval. 3 Open in new tabDownload slide Correlation analysis of cultivation efficiencies obtained by the MicroDrop and MPN techniques based on the full dataset. Filled circles (●) give pairs of data where MicroDrop values were significantly lower than MPN values. All other data pairs are given as open circles (○). Line indicates linear regression and dotted lines the 95% confidence interval. The significant differences between the results obtained with both techniques may either result from an underestimation of culturable bacteria by the MicroDrop technique and/or from an overestimation by the MPN method. The specific characteristic of the novel MicroDrop technique is the formation of minute droplets which harbor only one or a few bacterial cells. In theory, the exposure to high surface tension and shear forces during the formation of very small droplets could affect the bacterial cells and hence lead to the systematically lower cultivation efficiencies obtained in the MicroDrop experiments. However, the viability of Escherichia coli cells is not affected by this treatment [33]. Even the type 1 fimbriae of E. coli HB101 which are highly sensitive to mechanical forces are not disrupted by the passage through the nozzle of the capillary [33]. Therefore it appears more likely that the MPN method overestimates the number of culturable cells in natural samples. One reason for this positive bias may be that microorganisms occur in clumps [35] and that cells are dislodged only during handling of the sample [34]. However, the MicroDrop technique would also be effected by the presence of bacterial aggregates, albeit to a lesser extend, since most aggregates will be disintegrated during initial loading of the micropipet due to its small inner diameter. Most likely, the presence of bacterial aggregates represents a less likely reason for the systematic difference between MPN and MicroDrop data. An inherent tendency of the MPN technique for a positive bias has been noted earlier [34]. Further, indirect support for a pronounced positive bias of the MPN technique comes from the observation that numerically dominant bacteria could not be recovered in MPN series, even though high MPN values had indicated that the cultured bacteria must represent a large fraction of the natural microbial community [15–17,19]. 3.2 Diversity and phylogenetic affiliation of bacteria recovered with the different cultivation techniques Besides significant differences in cultivation efficiencies, it is also feasible that both cultivation techniques select different bacteria. Therefore bacteria from wells exhibiting growth in MicroDrop microtiter plates and bacteria from the highest positive dilutions of MPN series were subcultured and compared by DGGE of 16S rDNA fragments and subsequent sequencing. A total of 344 cultures obtained by the MicroDrop technique and 226 cultures generated by the MPN approach were analyzed. As an initial assessment of bacterial diversity, the ∼620-bp-long 16S rDNA fragments amplified from single microtiter wells were compared by DGGE fingerprinting. In most cases, single DNA bands were detected, indicating the presence of pure or highly enriched cultures in single wells (Fig. 4). Interestingly, cultures obtained by the MPN method yielded more than one melting type in 29% of all cases (Fig. 4B), while only 6% of those generated by the MicroDrop technique contained more than one fingerprint (Fig. 4A). Theoretically, one and the same bacterium may contain multiple rRNA operons and yield different fingerprints on DGGE gels [36]. In our case, however, one of the two fingerprints detected in some of the wells was also found as the sole fingerprint in other wells (Fig. 4). Therefore it appears more likely that different fingerprints represent different bacteria occurring together in one microtiter well. In conclusion, our results indicate that the MicroDrop technique is more suitable for the isolation of pure cultures than the MPN technique. 4 Open in new tabDownload slide Analysis of cultures by DGGE. A: Fingerprints from different wells exhibiting growth after inoculation with the MicroDrop technique. B: Fingerprints obtained from the highest positive dilutions of MPN series. Arrows indicate lanes (hence wells) containing bacteria with different 16S rDNA fingerprints. 4 Open in new tabDownload slide Analysis of cultures by DGGE. A: Fingerprints from different wells exhibiting growth after inoculation with the MicroDrop technique. B: Fingerprints obtained from the highest positive dilutions of MPN series. Arrows indicate lanes (hence wells) containing bacteria with different 16S rDNA fingerprints. Between 15 and 29 different melting types (positions of DNA bands on DGGE gels) were detected for each lake. The cumulative frequency of melting types was used to evaluate whether each culture collection represented the entire diversity which could theoretically be obtained by our cultivation method. Cumulative plots of melting types reached full saturation in all cases (Fig. 5). Therefore, additional cultivation attempts are highly unlikely to yield bacteria with novel 16S rDNA fingerprints. Irrespective of the technique used, the highest diversity of fingerprints was recovered from the bacterioplankton community of Zwischenahner Meer (Fig. 5A,B). However, the diversity of fingerprints obtained in MicroDrop cultures from Zwischenahner Meer was higher than that found in MPN cultures. 5 Open in new tabDownload slide Cumulative distribution frequency of melting types (DGGE bands) obtained after cultivation by (A) the MicroDrop or (B) the MPN technique. 5 Open in new tabDownload slide Cumulative distribution frequency of melting types (DGGE bands) obtained after cultivation by (A) the MicroDrop or (B) the MPN technique. The nine most frequent melting types as detected by the MicroDrop method were further analyzed. Depending on the frequency of a fingerprint, between four and 14 bands with the same melting behavior were excised and sequenced (Table 1, Fig. 6). For most melting types, several of the sequences obtained were identical. However, each melting type also included different sequences with identical melting behavior. Hence, the phylogenetic diversity obtained with the MicroDrop method is considerably higher than the diversity indicated by 16S rDNA fingerprinting. 1 Phylogenetic analysis of frequent melting types obtained by the MicroDrop and MPN techniquesa Melting type Frequency among all melting types (%) DGGE bands sequenced Identical sequences Sequences in tree Affiliation MicroDrop 1 8.7 6 3 , , ,wo38,wo51,zj97 β-Proteobacteria 2 11.0 7 2+3 (, ),(, , ),zj106,zj03 α-Proteobacteria 3 10.5 12 5 , , , , ,so06,so16,wo05,wo20,wo54 α-Proteobacteria zo43 β-Proteobacteria zo35 Flavobacteria 7 8.1 4 0 sj56,so41,so42,zj13 α-Proteobacteria 8 15.7 14 9 , , , , , , , , ,so03,so12 α-Proteobacteria zo44 α-Proteobacteria zj34 β-Proteobacteria wo11 Bacillales MPN 4 7.1 1 n.a. Mso1 α-Proteobacteria 5 7.1 3 0 Mwo2,Mzo1,Mzo3 β-Proteobacteria 6 9.3 1 n.a. Mzj3 α-Proteobacteria 8 10.6 2 2 , α-Proteobacteria 9 10.2 1 n.a. Msj1 α-Proteobacteria Melting type Frequency among all melting types (%) DGGE bands sequenced Identical sequences Sequences in tree Affiliation MicroDrop 1 8.7 6 3 , , ,wo38,wo51,zj97 β-Proteobacteria 2 11.0 7 2+3 (, ),(, , ),zj106,zj03 α-Proteobacteria 3 10.5 12 5 , , , , ,so06,so16,wo05,wo20,wo54 α-Proteobacteria zo43 β-Proteobacteria zo35 Flavobacteria 7 8.1 4 0 sj56,so41,so42,zj13 α-Proteobacteria 8 15.7 14 9 , , , , , , , , ,so03,so12 α-Proteobacteria zo44 α-Proteobacteria zj34 β-Proteobacteria wo11 Bacillales MPN 4 7.1 1 n.a. Mso1 α-Proteobacteria 5 7.1 3 0 Mwo2,Mzo1,Mzo3 β-Proteobacteria 6 9.3 1 n.a. Mzj3 α-Proteobacteria 8 10.6 2 2 , α-Proteobacteria 9 10.2 1 n.a. Msj1 α-Proteobacteria aIncreasing number of melting type corresponds to increasing melting stability in DGGE gels. Identical sequences of one melting type underlined and, if several different sequences were detected for the same melting type, are given in parentheses. n.a., not applicable. For strain designations compare legend to Fig. 6. Open in new tab 1 Phylogenetic analysis of frequent melting types obtained by the MicroDrop and MPN techniquesa Melting type Frequency among all melting types (%) DGGE bands sequenced Identical sequences Sequences in tree Affiliation MicroDrop 1 8.7 6 3 , , ,wo38,wo51,zj97 β-Proteobacteria 2 11.0 7 2+3 (, ),(, , ),zj106,zj03 α-Proteobacteria 3 10.5 12 5 , , , , ,so06,so16,wo05,wo20,wo54 α-Proteobacteria zo43 β-Proteobacteria zo35 Flavobacteria 7 8.1 4 0 sj56,so41,so42,zj13 α-Proteobacteria 8 15.7 14 9 , , , , , , , , ,so03,so12 α-Proteobacteria zo44 α-Proteobacteria zj34 β-Proteobacteria wo11 Bacillales MPN 4 7.1 1 n.a. Mso1 α-Proteobacteria 5 7.1 3 0 Mwo2,Mzo1,Mzo3 β-Proteobacteria 6 9.3 1 n.a. Mzj3 α-Proteobacteria 8 10.6 2 2 , α-Proteobacteria 9 10.2 1 n.a. Msj1 α-Proteobacteria Melting type Frequency among all melting types (%) DGGE bands sequenced Identical sequences Sequences in tree Affiliation MicroDrop 1 8.7 6 3 , , ,wo38,wo51,zj97 β-Proteobacteria 2 11.0 7 2+3 (, ),(, , ),zj106,zj03 α-Proteobacteria 3 10.5 12 5 , , , , ,so06,so16,wo05,wo20,wo54 α-Proteobacteria zo43 β-Proteobacteria zo35 Flavobacteria 7 8.1 4 0 sj56,so41,so42,zj13 α-Proteobacteria 8 15.7 14 9 , , , , , , , , ,so03,so12 α-Proteobacteria zo44 α-Proteobacteria zj34 β-Proteobacteria wo11 Bacillales MPN 4 7.1 1 n.a. Mso1 α-Proteobacteria 5 7.1 3 0 Mwo2,Mzo1,Mzo3 β-Proteobacteria 6 9.3 1 n.a. Mzj3 α-Proteobacteria 8 10.6 2 2 , α-Proteobacteria 9 10.2 1 n.a. Msj1 α-Proteobacteria aIncreasing number of melting type corresponds to increasing melting stability in DGGE gels. Identical sequences of one melting type underlined and, if several different sequences were detected for the same melting type, are given in parentheses. n.a., not applicable. For strain designations compare legend to Fig. 6. Open in new tab 6 Open in new tabDownload slide Open in new tabDownload slide Phylogenetic tree calculated by the maximum likelihood showing sequences from MPN (denoted by a capital M) and MicroDrop cultures which were obtained from Starnberger See (denoted with s), Walchensee (w) and Zwischenahner Meer (z), and their closest relatives. The letter j denotes samples obtained in July 2001, o denotes samples obtained in October 2001. A: Members of the α-subclass of the Proteobacteria. B: Members of five other phylogenetic groups. Designations in bold face represent sequences obtained in the present study. Sequences obtained by both, the MicroDrop and MPN technique shaded in gray. Scale bar indicates 10% sequence divergence. 6 Open in new tabDownload slide Open in new tabDownload slide Phylogenetic tree calculated by the maximum likelihood showing sequences from MPN (denoted by a capital M) and MicroDrop cultures which were obtained from Starnberger See (denoted with s), Walchensee (w) and Zwischenahner Meer (z), and their closest relatives. The letter j denotes samples obtained in July 2001, o denotes samples obtained in October 2001. A: Members of the α-subclass of the Proteobacteria. B: Members of five other phylogenetic groups. Designations in bold face represent sequences obtained in the present study. Sequences obtained by both, the MicroDrop and MPN technique shaded in gray. Scale bar indicates 10% sequence divergence. In parallel, several DNA bands of the five most frequent melting types of the MPN culture collection were also sequenced. Sequence comparison revealed that melting type no. 8 was the dominant melting type among both, the MicroDrop and the MPN cultures, and actually contained identical sequences (Table 1, Fig. 6A, shaded in gray). Furthermore, two other partial 16S rDNA sequences (zj03/Mzj1, Fig. 6A, and wj16/Mwj1, Fig. 6B) were detected in MicroDrop as well as in MPN cultures which had been inoculated with the same sample. This congruence between the dominant bacterial 16S rDNA sequences recovered suggests that both techniques select for similar groups of bacteria. It also suggests that the significantly lower culturability observed for the MicroDrop technique cannot be explained by a selection against a particular phylogenetic group of bacteria. The majority of the 112 sequences analyzed were affiliated with the α- and β-subclasses of the Proteobacteria. Fewer sequences belonged to either Flavobacteria, the Actinomycetales or Bacillales (Fig. 6). In particular, many of the sequences of α- and β-Proteobacteria and Flavobacteria were closely related to not-yet-cultured bacteria. Many of the sequences were only distantly related to their closest relative, showing a sequence similarity of less than 90% (Fig. 6). This indicates that the cultivation technique established in the present study is well suited to isolate novel phylotypes of freshwater planktonic bacteria. In several instances, identical partial sequences were recovered from more than one lake (Table 1). One of the sequences was detected in all three lakes on the same sampling date (so30,wo07,zo36), and was also identical to that of a numerically dominant uncultured β-Proteobacterium of the bacterioplankton assemblage in Zwischenahner Meer which had been described previously [18] (uncultured Zwischenahner Meer bacterium AF311985; Fig. 6B). This finding not only suggests that at least some of the freshwater planktonic bacteria occur widely distributed in lakes of different trophic status and physicochemical conditions, but also that some of the numerically important members of freshwater bacterioplankton can be cultured employing our MicroDrop technique. Similar to previous observations [15,17], cyclic AMP (cAMP) was the most effective signal compound and, when added in trace amounts, often resulted in significantly elevated cultivation efficiencies, especially in oligotrophic Walchensee (Fig. 2). Obviously, stimulation of bacterial growth by cAMP is not limited to marine [15] or eutrophic freshwater [17] habitats, but occurs in very different planktonic habitats. 3.3 Advantages of the MicroDrop technique In conclusion, the MicroDrop technique appears to be well suited for the generation of large numbers of numerically dominant culturable bacteria. DNA reassociation kinetics indicate that bacterioplankton communities consist of a high number of different species, estimated to amount to about 550 [18]. The MPN technique is less appropriate if the taxonomic composition of bacterioplankton communities is characterized by a high eveness, and hence consist of many different prokaryotes occurring at a similar but low frequency, since mere chance would dictate which isolate is obtained from the few highest positive dilutions available. In addition, bacteria dominant in natural bacterial communities may exhibit an especially low culturability in laboratory media, as indicated by recent attempts to cultivate planktonic actinobacteria [17]. A high eveness of bacterioplankton composition and a low frequency of culturable cells of numerically dominant planktonic bacteria make it mandatory to inoculate large numbers of parallels in order to obtain isolates of interest. In a recent approach [20], this was accomplished by diluting natural bacterioplankton samples and subsequently inoculating a large number of parallels by hand. This so-called ‘extinction culturing’ approach can be significantly accelerated and standardized by our MicroDrop technique which does not require initial dilution (a potential source of error) for most bacterioplankton samples, and permits the inoculation of 96 sample wells within less than 1 min. Acknowledgements Thanks are due to Frederic Gich, Karin Schubert and Martina Sterz for help with sampling and to Andrea Schlingloff and Heike Oetting for help with sequencing. This work was funded by the BMBF (Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie) to J. Overmann and H. Cypionka, Grant no. 0311949 and to J. Overmann, Grant no. BIOLOG/01LC0021. References [1] Amann R Ludwig W Schleifer K ( 1995 ) Phylogenetic identification and in situ detection of individual microbial cells without cultivation . 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TI - A novel approach for high throughput cultivation assays and the isolation of planktonic bacteria
JF - FEMS Microbiology Ecology
DO - 10.1016/S0168-6496(03)00133-8
DA - 2003-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-novel-approach-for-high-throughput-cultivation-assays-and-the-Yh0WPDHkfI
SP - 161
EP - 171
VL - 45
IS - 2
DP - DeepDyve
ER -