Isolation
and
Characterization
of
Uncultured
Freshwater
Bacterioplankton
from
Lake
Ekoln
and
Lake
Erken
through
Dilution-to-Extinction
Approach
and
Molecular
Analysis
Tools
Jiazhuo
Zhang
Degree project inapplied biotechnology, Master ofScience (2years), 2012 Examensarbete itillämpad bioteknik 30 hp tillmasterexamen, 2012
Biology Education Centre and Department ofEcology and Genetics, Limnology, Uppsala University Supervisor: Alexander Eiler
Abstract
Abstract
Abstract
Abstract
Not many of the abundant freshwater bacterial groups have a representative cultured isolate. In this master thesis project , some abundant bacterioplankton from two lakes (Lake Ekoln and Lake Erken) could be isolated by a dilution-to-extinction approach. Sterilized lake water which was obtained through an ultrafiltration system was used resembling a natural medium. Specific fragments of 16s rRNA of the isolates were amplified by universal bacterial primers (27f and 1492r, 341f and 805r.) for
genotyping against a freshwater sequence database and RDP training set (Version 7). A total of 33 isolates from the two lakes were taxonomically classified and revealed the isolation of typical and abundant freshwater bacteria. Original bacterial
community of Lake Ekoln was also analyzed by 16S rRNA clone library construction for diversity study. Phylogenetic trees were built through neighbor-joining method by Mega (Version 5) to reveal the evolutionary relationships among database entries, obtained isolates and clones.
Introduction
Introduction
Introduction
Introduction
Bacterioplankton is an irreplaceable component in aquatic ecosystems. Over 99% of the microbial species are uncultured yet, which means uncultured microorganisms compose the main part of microbial diversity on earth. Pure cultures of these
microorganisms have not been obtained through traditional culturing methods (Pace, 1997) since many strains do not grow in nutrient-rich (eutrophic) media. Trace elements, complex composition of dissolved organic carbon and microbial
interactions are other factors that affect the success of cult ivation. In this case, natural media is a good choice as the dynamic oligotrophic natural environment is difficult to mimic (Giovannoni and Stingl, 2007). Culturing the abundant and ecosystem-relevant microorganisms can help us to learn more about their biological and genetic features, and will further reveal their requirements and ecological roles (Zengler, 2009). At the
same time, metabolites of some organisms can be of interest in biotechnological applications. Thus, studies in this field are valuable no matter for academic research or industrial application.
Technology has been a key factor in the study of uncultured microorganism. Many molecular tools, such as RFLP (restriction fragment length polymorphism ),
CARD-FISH (catalyzed reporter deposition fluorescent in situ hybridization) and PCR (polymerase chain reaction), are commonly used for characterization over the last decade. Yet, the improvement of culturing approaches has attracted less interest. Combinative isolation techniques and new cultivation methods have been established for marine organisms (Giovannoni and Stingl, 2007). Button et al. (1993) developed dilution culture that used pristine seawater as media, and successfully isolated and cultured oligotrophic bacteria from the ocean. Media was prepared via membrane filtration and autoclaving. The cultures were incubated over three weeks in the dark to prevent autotrophy and photolysis. This technique selected abundant bacteria rather than nutrient-tolerant organisms (Button et al., 1993). Later, Connon and Giovannoni used 48-well microtiter plate to achieve high-throughput culturing, which was based on Button's idea. In their study, the inoculum was diluted to 1-5 cells/ml and
inoculated 1 ml/well. Abundant new marine isolates belonging to four lineages were obtained by this method (Connon and Giovannoni, 2002). Stinglet al. (2007) improved this technique to increase efficiency ofSAR11 screening. DMSP
(dimethylsulfoniopropionate) which is the substrate ofSAR11 strains was added in the media, and flow cytometry was applied as a fast and reliable tool for screening. A 24-well ultra-clean Teflon plate was used as culture vessel instead of microtiter plate. 5 ml of diluted inoculum were inoculated in each well, which allows more sampling according to variable lag phase and growth rate of bacteria. The cultivation time was prolonged over 4 weeks. Finally 17 newSAR11 strains were obtained with this improved method. (Stinglet al., 2007) In addition, they successfully used tangential flow filtration system in media preparation for the bacteria from Antarctic ice-covered lakes (Stingl et al., 2008). Future development focuses on designing optimum
culturing methods for different organisms (Hutalle-Schmelzeret al., 2010).
Common bacterioplankton in freshwater lakes belongs to three phyla:Phylum Actinobacteria, Phylum Bacteroidetes, Class Alphaproteobacteria and Class Betaproteobacteria of Phylum Proteobacteria. They account for more than 90% of the reported sequences in RDP (ribosomal database project) database. More than half of the bacteria in the surface water areActinobacteria which are gram-positive and have high G+C proportion in the genome. Most of the bacteria in this phylum are aerobic and work as decomposers in the ecosystem. Bacteroidetes are involved in organic matter degradation and aquatic food webs. Most of them are
chemoorganotrophs. Alphaproteobacteria also participate in degrading organic
compounds. Although they are distributed widely both in freshwater and seawater, we know little about them until now.Betaproteobacteria are the most well-studied group and second abundant bacteria in the surface lake water. The abundance of them is high in freshwater lakes and low in the ocean. They are fast-growing and nutrient-favoring. Gammaproteobacteria are abundant in saltwater. They are usually enterics of bacterial community in the lake. (Newtonet al., 2011) This is summarized in Table 1, which shows the classification and proportion of major bacterial phyla in freshwater lakes.
Table 1. Proportion of major phyla of freshwater lake bacteria in RDP database
Phylum Class Proportion in database
Full seq. Partial seq.
Actinobacteria - 58% 33% Bacteroidetes - 12% 17% Proteobacteria Alphaproteobacteria 6% 7% Betaproteobacteria 15% 23% Gammaproteobacteria 3% 5%
The aim of this project was to isolate uncultured bacteria from freshwater lakes. In order to succeed, I applied a modified approach based on Stinglet al. (2008).
Tangential flow ultrafiltration which combines minitan system with pellicon device was used to obtain natural media. This filtration-based sterilization procedure does not require autoclaving. It removes impurities physically, and will not change pH,
composition of organic matter and other chemical features of the lake water.
Dilution-to-extinction method was applied to acquire enrichments and pure cultures of abundant bacteria. The inoculum was diluted to approximately 1 cell/ml and then inoculated as 1 ml/well in the S-blocks. 2 or 4 weeks of static cultivation was performed at room temperature. Flow cytometry was applied for cell counting to achieve high-throughput screening (Stingl et al., 2007). Positive cultures were extracted by following a procedure proposed by Stinglet al. (2008). Then isolates were identified through PCR and sequencing. Phylogenetic analysis of 16s rRNA gave an overview of the phylogenetic relationship of the isolates with database entries and the original bacterial community of Lake Ekoln. Thus, this project provides a proof of principle and encourages for future attempts to isolate and culture novel freshwater lake bacteria using the established method.
Materials
Materials
Materials
Materials and
and
and
and Methods
Methods
Methods
Methods
As it was a microbial and molecular experiment, all the appliances were sterilized to prevent contamination.
Sampling Sampling SamplingSampling
Lake Ekoln and Lake Erken are large freshwater lakes in Uppland, Sweden. Lake Erken (Fig 1, 59o25' N, 18o15' E) is situated on the east shore of Sweden. pH value of
the lake water is around 8, and oxygen content is high. Water quality of this lake is good, although it is moderately eutrophic. (Hernández et al., 1999) Lake Ekoln (59o
45' N, 17o37' E) belongs to Lake Mälaren which is the third largest lake in Sweden.
River Fyrisån of Uppland flows into this lake. (Wikipedia, 2010-08-30) It has similar water color as Lake Erken.
Figure 1. Picture of Lake Erken (http://www.ebc.uu.se/Research/IEG/erken/)
Approximately 5 L of surface lake water were collected from each lake and stored in a plastic water tank. The water of Lake Ekoln was collected by Alexander Eiler on June 29th, 2011. And Lake Erken was sampled on June 14th, 2011.
Media Media
MediaMedia PreparationPreparationPreparationPreparation
Pellicon-Minitan Ultrafiltration System (Millipore Corporation) which is based on tangential flow filtration was used to filter original lake water to obtain natural medium. It is a combination of two filtration systems. The microporous sheets of minitan system (Φ=0.45 μm + 0.2 μm) removed most organisms and solid particles in the lake water. Then the permeate was further filtered by pellicon system, which eliminated all the organisms including viruses and macromolecular proteins whose molecular weights are above 50 kD. (Fig. 2) The filtrate was collected and divided into small volumes, and stored at 4oC.
Figure 2. Sketch map showing the workflow of Minitan-Pellicon Ultrafiltration System
150 ml and 200 ml of original water from Lake Ekoln were filtered by suction filtration (Φ=0.22 μm) respectively. The membrane filters were kept in small tubes and preserved at -80oC for DNA extraction of bacterial community.
Bacterial Bacterial
BacterialBacterial CountingCountingCountingCounting
Flow cytometer (CyFlow®Space, PARTEC Corporation) was used for bacterial
counting (Giorgio et al., 1996). Samples were prepared in 96-well plates as below: 200 μl of sample
2% filtered formaldehyde (final concentration) 25 μM of Syto 13 (working concentration)
Syto 13 is a fluorescent dye binding to DNA. The flow cytometer takes 50 μl of sample, cells pass through the optical system as a single line, then detectors capture those optical signals and convert them to electric signals. The value of bacterial concentration can be obtained then.
Dilution-to-Extinction Dilution-to-Extinction
Dilution-to-ExtinctionDilution-to-Extinction CulturingCulturingCulturingCulturing
key technique in this project.
Bacterial concentration of the lake water was measured three times by flow cytometer. The mean value was considered as initial concentration. Obtained natural media was used as diluent. Lake water was diluted to 1 cell/ml gradually by media. 1 ml of diluted lake water was inoculated to each well of a S-block. There were 6x96 cultures from each lake.
The cultures were cultivated statically at room temperature. After two weeks of cultivation, 250 μl of the culture in each well were transferred to a 96-well microtiter plate and preserved at -80oC for DNA extraction. Cultures from Lake Erken were
cultivated for four weeks. Then bacterial concentration of the cultures was measured with flow cytometer. Those cultures whose concentrations were higher than 105
cells/ml were considered as positive. Respective cultures that had been cryopreserved in microtiter plates were picked out for DNA extraction.
To preserve strains, 150 μl of positive culture were transferred to an Eppendorf tube and mixed with glycerol to a final concentration of 10%, which would protect cells from ice crystal. The tubes were put at -20oC for one hour before transferring to -80 oC for long term preservation.
DNA DNA
DNADNA ExtractionExtractionExtractionExtraction
DNeasy®Blood & Tissue Kit (Qiagen Corporation) was applied for DNA extraction.
The protocols were modified from the handbook of the kit as described below.
DNA extraction for cultures: 250 μl of culture were frozen and thawed (-80oC and 70 oC) for three cycles to lyse the cells. Adequate volumes of Proteinase K and Buffer AL
were added and samples were incubated at 70oC for 10 minutes to degrade membrane proteins. Then DNA was gradually purified with 99.9% ethanol, Buffer AW1 and Buffer AW2. The spin columns were dried in the incubator at 70oC for 3 minutes.
Finally, DNA was eluted with 100 μl of preheated Milli-Q water (70oC) and
preserved at -20oC for PCR.
DNA extraction for membrane filters (original lake community): One of the preserved filters was cut up and dipped in 250 μl of MQ water. Firstly the tube was incubated at 37oC for one hour. Then Proteinase K and Buffer AL were added. The tube was
incubated at 70oC for half an hour. 99.9% ethanol, Buffer AW1 and Buffer AW2 were
added respectively, and the supernatant was discarded after each centrifugation. The column was dried at 70oC for 10 minutes, then DNA was eluted twice by MQ water
to obtain a final volume of 200 μl. Then the tube was stored at -20oC for cloning.
PCR PCR PCRPCR
Taq DNA Polymerase recombinant (Invitrogen Corporation) and two pairs of primers were chosen and used for amplification (PCR machine: LifePro Thermal cycler, BT-TC-96LP, BIOER TECHNOLOGY Corporation). One pair was 27 forward-AGRGTTTGATCMTGGCTCAG and 1492 reverse- GGYTACCTTGTTACGACTT (Vergin et al., 1998), the other pair was 341 forward- CCTACGGGNGGCWGCAG and 805 reverse- GACTACHVGGGTATCTAATCC (Herlemannet al., 2011). Primers 27f and 1492r were used prior. If they performed badly, primers 341f and 805r were used subsequently. DNA concentration of a few samples was measured by nanodrop in order to estimate a rough range of the value and decide the volume of DNA
template added in PCR reactions. PCR reaction of 20 μl was adopted, which contains: 200 nM of Primer 27f and 1492r/ Or 400nM of Primer 341f and 805r
1 unit Taq DNA Polymerase 200 μM of dNTPs
Approximately 10 ng DNA template (~ 3 μl) 2 mM of MgCl2
2 μl of 10x PCR Buffer MQ water
According to the primers used in PCR reactions, protocol was switched as Table 2.
Table 2. PCR protocols for universal primers
Primer 27f and 1492r 341f and 805r
Temperature (oC ) Time (s) Temperature (oC ) Time (s)
Initialization 94 300 95 300 Denaturation 94 30 95 40 Annealing 50 30 58 40 Elongation 72 60 72 60 cycles 45 40 Final elongation 72 420 72 420 Final hold 4 -- 4
--If the quality of PCR products was not good enough after first round of amplification, a subsequent second round of PCR was performed. The protocol was modified to eliminate smear for second PCR: Annealing temperature would be risen 5oC and 10
cycles would be reduced according to Table 2. As the demand of sequencing, DNA quantity of 2 μl of PCR product should be no less than 15 ng.
Gel electrophoresis was performed as inspection method for PCR products. 2 μl of each PCR product and 0.5 μl of loading buffer were mixed and loaded on 1% agarose gel which was stained by red dye. 2 μl of Low DNA Mass Ladder (Invitrogen
Corporation) was loaded as marker. Electrophoresis was terminated after 30 minutes under the condition of 120 volt and 350 mA. Then the bands were examined in the UV chamber and DNA quantity was estimated by a program called "Gel Analyzer".
Cloning Cloning CloningCloning
Since filter from Lake Erken was not preserved at the beginning of this project, cloning was done only for the sample from Lake Ekoln following the protocol by
Eiler and Bertilsson (2004).
Selective LB plates and liquid LB medium were prepared at the beginning of this step. Kanamycin (50 μg/ml) was added when the medium had cooled down to 50oC. Then
the medium was stored at 4oC.
One of the cryopreserved membrane filters was taken out to extract DNA. Primers 341f and 805r were used for PCR amplification. Three replicates of 50 μl PCR reaction were prepared and 20 cycles of amplification were performed to minimize PCR bias (Polz and Cavanaugh, 1998). At first, DNA was precipitated for purification purpose. PCR products were combined and MQ water was added to make a total volume of 240 μl. Then 43.2 μl of 2 M sodium acetate were pipetted and well-mixed. Two volumes of 99.9% ethanol were added. The tube was incubated at -20oC for one
and a half hours and centrifuged at 13,200 rpm for 20 minutes. Supernatant was discarded and 750 μl of 70% ethanol was added. Another centrifugation of 10 minutes was performed. The precipitate was dried in the hood and then suspended in 30 μl of MQ water. Each 15 μl of the solution was loaded on the gel separately. After
electrophoresis, the bands were shown under UV light, and extra cted with Gel
extraction kit (Invitrogen Corporation). Microcentrifuge method in the handbook was applied here (QIAquick®Spin Handbook, P25).
TOPO TA cloning®kit (Invitrogen Corporation) was used then. Purified PCR product
has to be used within two days. SOC medium was prewarmed in 42oC water bath
before start. The clone reaction was composed of 3 μl of PCR product, 1 μl of salt solution, 0.5 μl of vector and 1.5 μl of MQ water. It was incubated at room
temperature for half an hour and then placed on ice. LB plates were prewarmed at 37
oC for 30 minutes. After that, 80 μl of X-gal (20 mg/ml) were spread on the surface of
each plate. Then the plates were held at 37oC until use. 25 μl of TOP10E.coli were thawed on ice and 2.5 μl of clone reaction were pipetted into the vial and mixed gently with the cells. The vial was incubated on ice for 30 minutes, then transferred to
42oC water bath for 30 seconds to heat-shock the cells, and placed on ice immediately.
250 μl of SOC medium which had cooled down to room temperature were added into the vial. It was shaken horizontally (about 200 rpm) at 35oC for one hour. Then 40 μl
and 80 μl of transformed cells were spread on prewarmed LB plates respectively. The plates were incubated at 37oC over night.
150 μl of liquid LB medium were pipetted to each well of a 96-well plate. White clones were picked and inoculated to each well. The plate was fixed on a shaker (about 200 rpm) and cultivated at 37oC over 24 hours. Then 75 μl of the culture in
each well was transferred to a new 96-well plate. To extract DNA, the plate was centrifuged at 4,000 rpm for half an hour and then centrifuged up-side-down for one minute at 200 rpm. 30 μl of MQ water was added to each well and the plate was heated at 98oC for 10 minutes. PCR reactions of 20 μl were prepared as below:
100 nM of Primer M13 forward and M13 reverse 1.25 unit Taq DNA Polymerase
200 μM of dNTPs 2 mM of MgCl2
2 μl of 10x PCR buffer 1 μl of DNA template MQ water
PCR protocol for M13 primer was adopted (Tab. 3). And then PCR products were quantified on the gel, too.
Table 3. PCR protocol for M13 primer
Primer M13f and M13r
Temperature (oC ) Time (s)
Initialization 95 60
Annealing 55 60 Elongation 72 120 cycles 25 Final elongation 72 300 Final hold 4 --DNA DNA
DNADNA SequencingSequencingSequencingSequencing andandandand PhylogeneticPhylogeneticPhylogeneticPhylogenetic AnalysisAnalysisAnalysisAnalysis
The PCR products were delivered to Uppsala Genome Center for Sanger sequencing. Prior to sequencing, PCR products had to be diluted at least 7 times to minimize the interference of excessive primers and reagents from the PCR reaction since
purification was not carried out. According to the result of quantification, PCR products of the isolates whose DNA concentrations were more than 15 ng/μl were diluted 7 times with MQ water while clones were diluted 100 times. According to the demand of Uppsala Genome Center, suitable volumes of the diluted PCR product were mixed with 4 pmol of respective forward primer, and MQ water was added to make a final volume of 18 μl.
The software "sequence scanner" (Applied Biosystems, USA) was used to check the quality of raw data signal of sequencing. Undefined nucleotides in the sequence were corrected manually. Obtained sequences were taxonomically classified by the
program "mothur". RDP training set (version 7, released on Nov. 20th, 2011) was
applied as template file. And another database "FWGG training set" (Sept. 9th, 2011)
(Newton et al., 2011) was also applied to see if the results were consistent or not. Reported sequences in NCBI database which have high similarities (≥97%) with the isolates were retrieved by BLASTn (Nucleotide Basic Local Alignment Search Tool) (Altschul et al., 1997). Then sequence alignments were done and phylogenetic trees were constructed with neighbor-joining method using the program "Mega" (Acinaset al., 1999).
Results
Results
Results
Results
Average bacterial concentration of original water from Lake Ekoln was about 8.8×105
cells/ml. The inoculum was diluted according to this value. Two weeks later, about 20% of the cultures were positive. After four weeks of cultivation, the proportion went up to approximately 40%. Then the number of positive cultures declined. After DNA extraction, the mean value of DNA concentration of the isolates was less than 3 ng/μl. Finally, 33 isolates were sequenced successfully: 13 came from Lake Ekoln and 20 were from Lake Erken.
The quality of raw data signal of sequencing was not so good due to the bad quality of PCR products. The valid sequences were often short, which brought more difficulties for identification. Most of them could only be classified to the genus level. Taxonomic affiliation determined by FWGG training set does not show much difference with the result identified with RDP database (Tab. 4).
Table 4. Taxonomic affiliation of the isolates determined with RDP training set
Habitat Code Phylum / Class Closest genus Similarity Length (bp)
Ekoln
K2-H8 Betaproteobacteria Limnohabitans 100% 810 K5-B10 Betaproteobacteria Alcaligenaceae 100% 837 K1-F9 Betaproteobacteria Polynucleobacter 100% 310 K5-A11 Betaproteobacteria Polynucleobacter 100% 747 K5-F6 Betaproteobacteria Polynucleobacter 100% 724 K2-A10 Bacteroidetes Flavobacterium 100% 264 K2-C12 Bacteroidetes Flavobacterium 100% 341 K2-D5 Bacteroidetes Flavobacterium 100% 801 K5-D7 Bacteroidetes Flavobacterium 100% 785
K5-E2 Gammaproteobacteria Vibrio 96% 557 K5-B11 Gammaproteobacteria Vibrio 100% 748 K5-D10 Gammaproteobacteria Vibrio 100% 666
Erken
R3-A6 Bacteroidetes Flavobacterium 100% 701 R2-B3 Alphaproteobacteria Rhodobacter 97% 625 R2-C6 Betaproteobacteria Polynucleobacter 100% 554 R2-E1 Betaproteobacteria Polynucleobacter 100% 789 R3-G3 Betaproteobacteria Polynucleobacter 100% 791 R2-C4 Betaproteobacteria Limnohabitans 100% 579 R2-D3 Betaproteobacteria Limnohabitans 100% 843 R2-D12 Betaproteobacteria Limnohabitans 100% 836 R2-G4 Betaproteobacteria Limnohabitans 100% 449 R3-A4 Betaproteobacteria Limnohabitans 100% 895 R3-E4 Betaproteobacteria Limnohabitans 100% 733 R2-D9 Betaproteobacteria Comamonadaceae 92% 502 R2-F9 Betaproteobacteria Polaromonas 100% 765
R1-D2 Gammaproteobacteria Vibrio 82% 421
R2-A3 Gammaproteobacteria Vibrio 98% 795
R3-B8 Gammaproteobacteria Vibrio 99% 823
R2-A5 Gammaproteobacteria Vibrio 100% 619 R2-B1 Gammaproteobacteria Vibrio 100% 791 R2-H2 Gammaproteobacteria Allomonas 96% 368 R4-A8 Gammaproteobacteria Pseudomonas 100% 774
We can see that no Actinobacteria was obtained and most isolates from both lakes belong to three major divisions:Bacteroidetes, Betaproteobacteria and
Gammaproteobacteria. For Lake Ekoln, each of them accounts for 1/3 of the sum. For Lake Erken, half of them are Betaproteobacteria, 1/3 are Gammaproteobacteria. And only one Bacteroidetes and one Alphaproteobacterium were acquired.
Phylogenetic trees were built respectively to show the phylogenetic distances between the isolates and some reported sequences. (Fig. 3 and Fig. 4)
Figure 3. Phylogenetic tree showing the relationship between the isolates from Lake Erken and closely related sequences(bootstrap resampling: 1000; cut-off value: 50%)
Figure 4. Phylogenetic tree showing the relationship between the isolates from Lake Ekoln and closely related sequences(bootstrap resampling: 1000; cut-off value: 50%)
Clone library of Lake Ekoln was constructed by blue-white screen of cloning. White clones were picked from the plate with fewer clones. There were only a few blue clones growing on the selective plates. A nice batch of amplification was acquired at the end of cloning. Molecular size of PCR products was checked on the gel through comparing with DNA ladder. Taxonomic classification of the clones was also performed using the RDP database (Tab. 5).
Table 5. Summary of taxonomic classification of the clone library from Lake Ekoln
Tax. level Rank ID Taxonomy Daughter levels Total
0 0 Root 1 0 1 0.2 Bacteria 3 95 2 0.2.2 "Actinobacteria" 1 70 3 0.2.2.1 Actinobacteria 1 70 4 0.2.2.1.2 Actinomycetales 2 70 5 0.2.2.1.2.19 Intrasporangiaceae 1 69 6 0.2.2.1.2.19.18 Terracoccus 0 69 5 0.2.2.1.2.22 Kineosporiaceae 1 1 6 0.2.2.1.2.22.1 Angustibacter 0 1 2 0.2.5 "Bacteroidetes" 1 14 3 0.2.5.3 "Sphingobacteria" 1 14 4 0.2.5.3.1 "Sphingobacteriales" 2 14 5 0.2.5.3.1.5 Chitinophagaceae 1 13 6 0.2.5.3.1.5.14 Sediminibacterium 0 13 5 0.2.5.3.1.6 Cytophagaceae 1 1 6 0.2.5.3.1.6.2 Arcicella 0 1 2 0.2.21 "Proteobacteria" 1 11 3 0.2.21.3 Betaproteobacteria 1 11 4 0.2.21.3.1 Burkholderiales 1 11 5 0.2.21.3.1.2 Burkholderiaceae 1 11 6 0.2.21.3.1.2.7 Polynucleobacter 0 11
The clone library does not show high diversity of bacterial community in lake water with very atypical community composition for a freshwater lake. Reasons for this are unknown. Actinobacteria account for 74% of the sum. All of them are members of tribe acI-B4. 11% of the clones belong to tribe PnecD of Betaproteobacteria, and the
rest of them have high similarities with Family Chitinophagaceae of Bacteroidetes. (Fig. 5) These taxa are very common in freshwater lakes, but many more typical freshwater taxa were expected.
Figure 5. Taxonomic distribution of the clone library from Lake Ekoln
And Figure 6 shows the phylogenetic relationship between the clones and isolates from Lake Ekoln and some reported sequences retrieved by BLASTn.
N R 0 26 41 2. 1| T er ra co cc us lu te us s tra in D S M 4 42 67 N R 042 185. 1| In trasp or angi um calvu m DS M 430 43 s train : D SM 4 3043 N R 0 2580 5.1| J anib acte r m elon is stra in C M21 04 H4 H8 G3 G4G1H1 E8F8 E6G10 E5F12 H11 E3 H3 D12 F5 D11 E10 D 8 F6 D 6 F4 D 4 F2 C 12 D 1 G2 C 7 C 10 C 6 E7 C 5 E2 C4 E1 C 3 C 8 B8 D 7 B7 B10 B6 E9 H 2 B4 G5 B1 H 7 A12 D9 A10 H9 A9 B11 A8 B9 H 6 A7 D 10 A6 C 1 A5 F 10 A 4 H 5 A3 G 6 A 2 H 1 2 N R 0 43 46 5.1 | D em eq uin a a estu ar ii s train J C 20 54 IM S N U 14 02 7 K CT C 99 19 J C M 12 12 3 99 D 2 G8 F 3 G 7 NR 044 576. 1| P aras egitib acte r luo jiens is stra in RH Y L-37 B5 F7 C9 D5 C11 E11 G9 D3 G11 99 NR 0290 00.1| Arc icella aqua tica s train NO -502 NR 0435 54.1| Flec tobac illus l acus stra in CL -GP7 9 NR 0427 07.1| Arcice lla ro sea s train : TW 5 86 B12 99 NR 0 4458 1.1| F lavob acter ium ch unga ngen se str ain CJ 7 NR 0 4429 2.1| F lavob acter ium re sisten s stra in B D-b365 95 K2-C 12 NR 02 5923 .1| Fla voba cteriu m hib ernum strain ATCC 5146 8 NR 04 1057.1| F lavob acterium frigidim aris s train K UC-1 N R 04 2470.1| F lavobac terium aquidure nse stra in : WB 1 .1-56 K5-D 7 K2-D 5 K2-A10 65 93 98 74 83 N R 043 858.1| Vibrio sinaloen sis strain C AIM 7 97 K5-E2 K5-B11 K5-D10 N R 025478.1 | Vibrio xuii str ain R-15052 N R 025477.1| V
ibrio brasiliensis strain LM G 20546 N R 026129.1| Vibrio tubiash
ii strain Milford 74 N R 025491.1| Vibrio hepatarius strain
LM G 20362 N R 037067.1| Vibrio furnissii strain 9119-82
K5-B7 N R 025476.1| Vibrio neptunius strain LM G 20536
N R 036790.1| Vibrio fluvialis strain VL 5125
99
N R 025685.1| Achromobacter insolitus strain LM G 6003 N R 044925.1| Achromobacter xylosoxidans strain : D
SM 10346 N R 041769.1| Bordetella avium stra
in ATCC 35086 N R 025686.1| Achromobacte r spanius strain LMG 5911 N R 042021.1| Achrom obacter denitrificans s train DSM 30026 89 N R 0448 02.1| Castellaniella d enitrificans strain N KNTAU K5-B10 89 95 N R 025112.1 | Pigmentipha ga kullae strain K24 96 K2-H8 N R 0 28713.1| C urvibacter d elicatus stra in 146 N R 02865 5.1| C urvibacter grac ilis strain 7-1 NR 0 37133.1| O xalicibac terium fla vum strain TA 17 NR 042382 .1| P olynucle oba cter nece ssarius subsp. a sym bioticus Q LW -P1DM WA-1 strain Q LW -P1DM W A-1 NR 042555 .1| P olynucle obacter nec essariu s su bsp. nec essariu s 65 K1-FK5-A191 95 K5-F 6E4 F9F11 A11B2C2 E 12B3 H 10F1 G 12 91 84 81 54 91 74 87 50 99
Figure 6. Phylogenetic tree showing the relationship between clones and isolates from Lake Ekoln and closely related sequences(bootstrap resampling: 500; cut-off value: 50%)
Discussion
Discussion
Discussion
Discussion
The soleAlphaproteobacterium was isolated from Lake Erken. Members of Genus Rhodobacter are phototrophic and gram-negative. No pathogenic strains in this genus are known. They are not often obtained in freshwater lakes. Although
Gammaproteobacteria appear scarcely in freshwater, a member of Genus
Pseudomonas was obtained from Lake Erken. The isolates of Bacteroidetes from both lakes areFlavobacteria. Members of Flavo lineage are favored for organic matter and phytoplankton blooms. Except one isolate of minor genus Polaromonas obtained from Lake Erken, all isolates of Betaproteobacteria from both lakes are members of tribes PnecB, PnecD, Lhab-A1, Lhab-A2, Lhab-A3 and betIII-A1, which are considered as typical and very common freshwater bacteria. Tribe PnecB and Clade betI-A are typically more abundant under neutral to alkaline conditions. And PnecB shows obvious seasonal dynamics and its abundance declines as the depth increases. (Newton et al., 2011)
ManyGammaproteobacteria were obtained while almost no Actinobacteria and Alphaproteobacteria were isolated. Previous studies have suggested that
Actinobacteria are difficult to isolate (Newton et al., 2011), whereas
Betaproteobacteria and Bacteroidetes seem to be easier to acquire . The potential reason probably is that the cell walls of Actinobacteria are more difficult to lyse. A curiosity was that there were so many members of theGenus Vibrio which are very uncommon in the studied systems. I suggest that they were the result of a
contamination during the isolation experiment. Another explanation for this bias could be an introduction via used PCR reagents left by someone else, unclean appliances or careless operations.
Clones obtained by library construction from Lake Ekoln consisted of tribe Flecto and Family Chitinophagaceae of Bacteroidetes, tribe PnecD of Betaproteobacteria and tribe acI-B4 ofActinobacteria. acI lineage is prevalent in the upper water of lakes
while clade acI-B is often found in humic lakes (low pH) (Newton et al., 2011). Strains inFamily Chitinophagaceae are also humic matter-favoring which participate in relative degradation (Hutalle-Schmelzer et al., 2010). Tribe Flecto is filamentous and abundant under high grazing pressure. It seems to exist frequently in acidic lakes, too. And members of PnecD are ultramicrobacteria. (Newtonet al., 2011)
Very slight overlap exists between related isolates and clone library. Both the diversity of clone library and the number of isolates are too low and with deeper coverage which might change. Although the proportion of their distributions is consistent with Newton's study, acI-B4 and PnecD are not the most abundant tribes in freshwater lakes (Newtonet al., 2011). Other tribes like LD12, acI-B1 to B3 and acI-A1 are usually more abundant in Lake Ekoln. Maybe the primer 341f used in cloning, which has an additional GC-clamp, has higher affinity to these minor sequences. Or this sample was really special.
In this project, approximately 1,100 cultures from the two lakes were cultivated, and 340 cultures of them were positive, but only 33 isolates were sequenced successfully after DNA extraction and amplification. Except the bad quality of PCR products, a few cultures which contained more than one taxon would also result in overlapping signals of sequencing. But the main problem of this project was PCR performance. Different enzymes were tested and PCR protocols were optimized. New primers were used in case of the degradation of DNA templates. And more amounts of template were added in PCR reactions. In spite of that, there were still a lot of isolates which could not be amplified, or the quantity of their PCR products could not meet the demands of sequencing facility. Even after a second round of PCR, the bands were still weak. 3% DMSO (Dimethyl sulfoxide) in PCR reactions seemed to improve PCR performance and can be helpful in sequencing (Frackman et al,. 1998).
The bad performance of PCR was probably caused by many reasons: Contamination, ethanol residue of DNA extraction, bad quality of DNA template, difficulty of lysing
cells or applicability of primers for different cells (Stinglet al., 2008; Connon and Giovannoni, 2002). In this case, normative aseptic operations and prolongation of drying time for ethanol are needed. The key solution is to increase the concentration of DNA template. The experiment revealed that two weeks of cultivation were not long enough as these bacteria grew slowly in natural medium. Cultivation periods of over 4 weeks have been reported previously (Stinglet al., 2007). Adding more DNA templates in PCR reactions is another choice. Declining the volume of elution water in DNA extraction or using high pH eluent instead of MQ water is also helpful. There is still work to be done to improve DNA extraction and amplification from low cell number samples to establish a high-throughput culturing platform.
Moreover, comparing to Stingl's method, the cultures of mine were not cultivated in the darkness, and the initial concentration of inoculum was extremely low (Stingl et al., 2008). As the growth of some microorganisms depends on microbial interactions, these factors are the potential reasons for the low yield (Giovannoni and Stingl, 2007).
Acknowledgments
Acknowledgments
Acknowledgments
Acknowledgments
First of all, I'd like to thank my supervisor Alexander Eiler, for his encouragement, patience and expert advice. Then I have to thank Professor Stefan Bertilsson who gave me the chance to work in his group. Moreover, I want to express my gratitude to the PhD students in Limnology Department, especially Ina Severin who gave me many advices on my experiment, Hannes Peter who trained me to use the flow cytometer, Mercè Berga who ordered the reagents for me, Monica Ricao who guided me at the beginning of the project , and Torsten Jeske who acted as opponent of my final presentation. And I also have to thank the coordinator Anna-Kristina Brunberg who solved many problems for me. Finally, thank all the people who gave support throughout this project.
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