MÄLARDALENS HÖGSKOLA
School of Sustainable Development of Society and Technology
Optimization of biomolecular techniques for
detection of nitrite-oxidizing bacteria
Sara Sydkull
Degree project 15.0 ECTS Eskilstuna 2009 Supervisor: Professor Carl Påhlson
Abstract
Nitrification is a natural occurring, oxidative process which is essential for plants´ ability to take up nitrogen in the form of nitrate. The oxidation is divided into two steps. First ammonia is oxidized to nitrite by ammonia-oxidizing bacteria (AOB) or archaea (AOA) and then the nitrite is further oxidized to nitrate by nitrite-oxidizing bacteria (NOB). The enzyme used by NOB for the oxidation is nitrite oxidoreductase (nxr). One of few bacteria that catalyze this reaction is Nitrobacter sp.
The purpose of this study has been to optimize the detection of Nitrobacter in samples of activated sludge from municipal wastewater treatment plants (WWTPs) in Eskilstuna and Västerås (Sweden). This was done by PCR, cloning and sequencing. Primers used were nor F/nor R that are specific for the functional gene encoding nxr. This optimization has been compared to a different PCR-system where nor F/nor R were exchanged for another
primerpair consisting of a 16S rDNA-primer (NIT3), which was specific for Nitrobacter and a universal 16S-primer (U2, Rit388). In addition to this, a semi quantitative analyze has also been conducted.
The result of the study was two PCR-programs, one optimized for each set of sludge samples. The quantitative analysis showed that the concentration Nitrobacter in the sludge samples was approximately the same as a pure culture, which was used as a positive control and contained ~104 CFU/ml.
Cloning and sequencing revealed the presence of 3 different Nitrobacter. Surprisingly half of the clones from one of the Västerås samples, taken in December, were most likely
Methylibium petroleiphilum. The matter of fact that we were able to detect this bacterium with primers specifically designed for Nitrobacter made this discovery very interesting. With NIT3/U2 Methylocella sp. was also detected in samples from Västerås, which confirmed the presence of methylotrophic bacteria in the Västerås samples.
Contents
Background………4
Introduction………5
Materials and methods………...6
Materials………6
Chemicals……….6
Bacterial type strain………..6
Primers………..6
Instruments………7
Methods………8
Preparations………8
Optimization of Mg2+-concentration………...8
Optimization of annealing temperature in 1st programme………...8
Adjustment of annealing temperature for February samples………...9
Optimized PCR of sludge samples……….9
Cloning and sequencing……….9
PCR with primers NIT3/U2………...9
Results………10
Optimal annealing temperature for 1st programme………...10
Optimized PCR for December samples………10
Optimized PCR for February samples………..10
Quantification of Nitrobacter winogradskyi……….11
PCR with primers NIT3/U2……….12
Discussion………...12
Acknowledgements………15
References………..16
Appendix 1
Recipes
LB-media TBE-buffer
Media for pure culture
Appendix 2
4
Background
In order to perform a qualitative/quantitative analysis of an environmental sample, in this case a sludge sample, one must first consider the factors affecting the performance of the analysis, e.g. organisms, substances and foreign DNA which interfere with the detection of the
microorganisms one wishes to study. An optimization of the detection method is therefore often required and this is also the underlying reason for the emergence of this work. This thesis is a part of a scientific study which aims to explore the diversity of nitrifying bacteria in the activated sludge from municipal wastewater treatment plants (WWTPs) in Eskilstuna and Västerås (Sweden).
5
Introduction
Chemolithoautotrophic bacteria are bacteria that derive energy from oxidizing inorganic compounds. Good examples of these bacteria are nitrifying bacteria that reduce nitrogen compounds.
Nitrification is an important part of the natural nitrogen cycle in which ammonia is converted into nitrate. The oxidation itself is a two-step process. The first step involves the oxidation of ammonia into nitrite by ammonia-oxidizing bacteria (AOB) or archaea (AOA) and the second involves further oxidization of nitrite into nitrate by nitrite-oxidizing bacteria (NOB). NOB consists of the genuses Nitrobacter, Nitrococcus, Nitrospina and Nitrospira. [1] The enzyme used by NOB is nitrite oxidoreductase (nxr) which can be found in the complex membrane system of the organisms. The abundance of nitrifying bacteria can be seen in soil and aquatic environments where protein degradation and thus higher ammonia levels are common. A typical habitat is therefore water purification plants. In these plants the water is often being purified in three steps. The second step of the purification involves oxidation of organic and inorganic material by microorganisms in the sludge. This is the most important step and it plays a significant role in the quality of the purified water. [2]
The main purpose of this study is to optimize the detection of Nitrobacter in sludge samples via PCR with primers that are specific for the gene nitrite oxidoreductase, nor F/nor R. The primer pair specifically hybridizes to the α-subunit of the gene and gives a 322 bp large fragment. This optimization is being compared to another system where nor F/nor R are exchanged for a Nitrobacter-specific 16S rDNA-primer (NIT3) and a universal 16S-primer (U2, Rit388). This primerpair amplifies a gene fragment of the size 633 bp. A semi
6
Materials and methods
Materials
Chemicals • Agarose • Ethanol 70 %, 100 % • EDTA 125 mM • MgCl2 25 mM • LB-media (appendix 1) • TBE-buffer (appendix 2)• 100 bp DNA ladder PLUS, Fermentas life sciences
(100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000 bp) • Platinum® PCR Supermix, Invitrogen
• PureTaq Ready-To-Go™ PCR Beads, GE Healthcare
Bacterial type strain
• Nitrobacter winogradskyi (DSMZ)
Primers
7
Table 1. Primers used for PCR-analysis of Nitrobacter in samples of activated sludge from WWTPs in Eskilstuna and Västerås.
Primer Sequence (5´ 3 ´)
Concentration (OD)
F1norA CAG ACC GAC GTG TGC GAA AG 2
R1norA TCY* ACA AGG AAC GGA AGG TC 2
NIT3 CCT GTG CTC CAT GCT CCG 1,5
U2, Rit388
CCA (A/G)AC TCC TAC GG(A/G) AGG CAG
C 2
M13 F GTA AAA CGA CGG CCA G 1,5
M13 R CAG GAA ACA GCT ATG AC 1,5
T7 F TAA TAC GAC TCA CTA TAG 1
*Y= 50/50 T/C
Instruments
• PCR Express Thermal Cycler, Hybaid Limited & T3 Thermocycler, Whatman, Biometra
8
Methods
Preparations
A pure culture of Nitrobacter winogradskyi was purchased from German Resource Center for biological material (DSMZ) GmbH, Germany. Cultivation was performed aerobically at 30
o
C. For details about media, see appendix 1.
Samples of activated sludge were collected from municipal wastewater treatment plants (WWTPs) in Eskilstuna and Västerås (Sweden), in December 2008 and February 2009. The sludge samples were taken from the aeration basins. Extractions of DNA were made with DNeasy® Blood and Tissue Kit (Qiagen) according to the manual for Gram-positive bacteria. Design of primers for PCR was based on previous studies of nxr-sequences of different Nitrobacter. [1] Primers were purchased from Invitrogen.
All PCR-reactions were carried out in tubes with the final volume 25 µl, whereof 1 µl template and 1 µl of each primer.
Optimization of Mg2+-concentration
Two dilution series were made in order to optimize the Mg2+-concentration for two different DNA polymerases. The template was Nitrobacter winogradskyi. Three micro-tubes were prepared with 0, 1 and 2 µl 25 mM MgCl2 respectively. One dilution serie was run with PCR
Platinum® Supermix and the other with PureTaq PCR-beads. The programme was as follows: 94 oC for 5 min followed by 35 cycles of 94 oC for30 s, 48 oC for 30 s and 72 oC for 45 s and a final elongation step of 72 oC for 10 min.
Optimization of annealing temperature in 1st programme
In order to get strong amplification a nested PCR was performed. For optimization of the annealing temperature in the first programme the pure culture was used as a template. Platinum® Supermix was used and the programme was the same as the one used for Mg2+ -optimization, with the only exception being a gradient of the annealing temperature between 47,7-59,7 oC according to table 2.
Table 2. PCR-tubes and their respective annealing temperature used for determining the optimal annealing temperature in the first PCR-programme.
Tube 1 2 3 4 5 6 7 8
Annealing
9
Adjustment of annealing temperature for February samples
Samples taken in February 2009 required an additional adjustment of the optimized PCR-programmes. By lowering the annealing temperature in the second programme by one degree the specific target sequence amplification was increased.
Optimized PCR of sludge samples
Optimized programmes were applied on all sludge samples. The first program was run with Platinum® Supermix. 25 µl of the product was transferred to new tubes with PureTaq PCR beads and thereafter the second program was run.
The optimized programmes were used for the semi-quantitative analysis of Nitrobacter winogradskyi. This was done by making a dilution serie of the pure culture in the range 1/10-1/106. As a comparison a similar dilution was made of one of the sludge samples.
Cloning and sequencing
Cloning was performed with TOPO TA Cloning® Kit for sequencing, pCR 2.1-TOPO Vector (Invitrogen). The clones were screened by PCR with primers M13 F/M13 R. Sequencing was performed with Thermo Sequenase™ Primer Cycle Sequencing Kit (GE Healthcare),
according to the manufacturer’s instructions.
PCR with primers NIT3/U2
PCR was conducted on all sludge samples as follows: 30 cycles of 94 oC for 30 s, 65oC for 30 s and 72 oC for 1 min and a final elongation step of 72 oC for 10 min. 25 µl of the product was transferred to new tubes with PureTaq PCR beads and thereafter another 30 cycles were run with the same programme.
Ligation of the PCR-product with vector pJET 1.2/blunt was done with CloneJET™ PCR Cloning Kit, #1239 (Fermentas), according to the manual. Transformation was performed with One Shot TOP10 Chemically Competent E.coli (Invitrogen).
Primers pJET forward/reverse were used for screening of selected clones and for the sequencing reaction, which was performed accordingly to BigDye® Terminator v3.1 Cycle sequencing Kit (Applied Biosystems).
10
Results
Optimal annealing temperature for 1st programme
The optimal temperature was determined to 55 oC.
Optimized PCR for December samples
Programme 1: 94 oC for 5 min followed by 35 cycles of 94 oC for 30 s, 55 oC for 30 s and 72
o
C for 45 s and thereafter a final elongation step of 72 oC for 10 min.
Programme 2: 35 cycles of 94 oC for 30 s, 58 oC for 45 s and 72 oC for 45 s and thereafter a final elongation step of 72 oC for 5 min.
Optimized PCR for February samples
Programme 1: 94 oC for 5 min followed by 35 cycles of 94 oC for30 s, 55 oC for 30 s and 72
o
C for 45 s and thereafter a final elongation step of 72 oC for 10 min.
Programme 2: 35 cycles of 94 oC for30 s, 57 oC for 45 s and 72 oC for 45 s and thereafter a final elongation step of 72 oC for 5 min.
The result of the optimized programmes can be seen in figure 1 and 2.
Figure 1. Optimized PCR of sludge samples taken in December 2008. The figure shows: Well 1) 100 bp size marker 2) Negative control 3-4) Positive controls 5-8) PCR-products with DNA-extracted sludge samples as template.
11 Figure 2. Optimized PCR of sludge samples taken in February 2009. The figure shows:
Well 1) 100 bp size marker 2) Negative control 3) Positive control 4-9) PCR-products with DNA-extracted sludge samples as template.
Quantification of Nitrobacter winogradskyi
The quantification showed that the concentration Nitrobacter winogradskyi in the pure culture was similar to that in the sludge sample, since amplification could be seen in the same number of dilution steps in both series, clearly illustrated by figure 3. Since the pure culture does not grow on solid media, a CFU determination was done by microscopy of a known volume and estimated to 104 CFU/ml.
Figure 3. Quantification of Nitrobacter winogradskyi with PCR. Two dilution series were made, with the highest concentration at the far left of the figure and declining to the right. The uppermost serie shows a dilution of Nitrobacter winogradskyi and the lower a dilution of a DNA-extracted sludge sample.
12
PCR with primers NIT3/U2
Obtained PCR-products can be seen in figure 4.
Figure 4. Result of PCR with primers NIT3/U2. The figure shows: Well 1) 100 bp size marker 2, 8) Negative controls 3, 9) Positive controls 4-7) DNA-extractions of sludge samples from dec-08 10-13) DNA-extractions of sludge samples from feb-09
Discussion
This study has made me realize the danger of contamination when conducting PCR,
especially the type (nested) used here. Transferring the products from the first program to new tubes posed the greatest risk for introducing contaminations to a so far closed system. PCR is a very sensitive method which can detect one single molecule of DNA in a sample. Therefore it was very important to work in a fume hood with clean instruments and to separate the work with PCR-products from the work with start material. To further reduce the risk of
contamination, by decreasing the use of pipettes, PureTaq PCR-beads were used instead of Platinum® Supermix in the second programme. According to the manual for PureTaq PCR-beads, unspecific amplification can be obtained when the number of cycles exceeds 35. It also states that the number of cycles should be between 20-40 cycles. [3] In this study 70 cycles were run per sample, which enhances the understanding of how easily unwanted
PCR-13 products could be obtained. Several of the attempts had to be remade because of
contaminations.
In a previous study primers nor F/nor R were used for detection of Nitrobacter in soil samples. [1] The same program was applied on selected sludge samples and gave the desired result, although the amplification resulted in relatively weak products. (Figure 1, appendix 2)
When the optimized December programmes were applied on the February samples it resulted in undesired products. This suggests that there were differences in the culture or in the
composition of the sample which interfered with the hybridization. Various parameters in the December programmes were then changed, one at a time, in order to get more specific amplification. First the number of cycles was changed in the second programme, since this programme was the one responsible for the emergence of unspecific products. Thereafter the annealing temperature was changed by lowering it by one degree in the first and then the second programme. Eventually a lower annealing temperature in combination with a lower number of cycles in the second programme was tested. A lower annealing temperature in the second programme proved to give the best result. The optimized February programmes gave, unlike the December programmes, an extra band apart from the desired. This band could be observed for all samples, including the positive control. Further work could include
optimization of the programme so that only one band is obtained. (Figure 2, appendix 2) Our quantitative analysis showed that dilution of the sludge sample gave increased
background amplification as compared to the pure culture. This confirmed that the matrixes of the sludge samples were more complex and affected the hybridization of the primers. The estimated concentration in the sludge samples should therefore be considered a minimum. Some of the clones that were obtained with nor F/nor R were sent to Karolinska Institutet (Stockholm, Sweden) for sequencing and phylogenetic analysis. A total of 80 clones were sent. The result of this analysis showed that the sludge samples contained gene sequences showing most similarity to the genomes of Nitrobacter winogradskyi, Nitrobacter
hamburgensis and Nitrobacter alkalicus. To our great surprise, half of the sequenced clones from Västerås were most likely Methylibium petroleiphilum, a bacterium which was not found in Eskilstuna.
Methylibium petroleiphilum is a Gram-negative that can reduce nitrate to nitrite, but not oxidize nitrite. It belongs to the Betaproteobacteria and can use e.g. methanol, ethanol, toluene and benzene as sole carbon sources. M. petroleiphilum can completely degrade methyl tert-butyl ether (MTBE), which is an important ability that can be used for MTBE-biodegradation of contaminated groundwater. [4]
14 The primers NIT3/U2 were chosen after testing the combinations NIT3/U2, NIT3/U3 and NIT3/U4 as potential primerpairs. NIT3/U2 was the only pair to give amplification and was chosen on this basis. After cloning, screening showed that all clones had the wrong fragment in the vector. Instead of the expected fragment of 835 bp, a fragment of 200 bp was found. After extracting the DNA-fragment from the electrophoresis gel a new cloning was made, but this did not help. Other actions included increasing the elongation time in the screening programme and trying a new cloning with longer incubation time for ligation and
transformation. None of these actions improved the result. Finally we tried out a new cloning kit (Fermentas). This kit gave numerous clones and screening also showed more clones with the right fragment than with Invitrogens kit.
Alignment analysis showed Clostridium sp. in both Eskilstuna- and Västerås samples and Methylocella sp. in Västerås samples. Cloning of the small fragment, approx. 200 bp, shows after sequencing to be some type of microsatellite-DNA. We chose not to continue looking for the origins of this fragment since this could be too time-consuming.
Methylocella sp. is a group methane oxidizing bacteria which can use e.g. methane and methanol as carbon sources. [5] The presence of methylotrophic bacteria in the Västerås samples is believed to be associated with the methanol that the WWTP uses as carbon source. When comparing the two PCR-systems one can see that they give the same result, which is the presence of methylotrophic bacteria in the sludge samples from Västerås. With NIT3/U2 Clostridium sp. could also be found in both Eskilstuna- and Västerås samples. In order to present a more comprehensive picture of bacteria that could be detected with this primerpair, more clones need to be analyzed. Finally, since NIT3 hybridized to other genera than
Nitrobacter the primer was not as specific as could theoretically be assumed.
The genome of a Nitrobacter representative, namely Nitrobacter hamburgensis and the genome of Methylibium petroleiphilum were compared with the database KEGG
(http://www.genome.jp). This comparison showed sequence similarities between their nitrate reductases. In turn, when running the nitrate reductase of N. hamburgensis through BLAST it shows 90 % nucleotide similarity with nxr at specific sites. This could be an explanation as to why nor F/nor R could hybridize to other genera than Nitrobacter. Further inquiries reveal that genetic studies have shown that nxr and the two nitrate reductases in E.coli (NRA and NRZ) are similar both structurally and functionally. Moreover, the α-subunit in nxr shows
remarkable sequence similarity to the α-subunits in E.colis nitrate reductase. [6] A hypothesis could be that N. hamburgensis and M. petroleiphilum have a common evolutionary origin.
15
Acknowledgements
Working on this degree project has been a valuable experience for me. I feel that I have grown both as a human being and as a student. My newly gained knowledge is something I will carry with me for the rest of my life and for this I have two persons to thank; my supervisor Carl Påhlson, an unlimited source of knowledge and a fantastic person whom I respect and PhD student Adrian Rodriguez, a great and admirable man who adopted the role as my teacher, 2nd supervisor and above all colleague, for which I am very thankful.
16
References
1. Franck Poly, Sophie Wertz, Elisabeth Brothier and Valerie Degrange; First
exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA; Federation of European Microbiological Societies (FEMS) microbiology ecology; 2008, vol. 63, pages 132-140
2. Michael T. Madigan, John M. Martinko; Brock Biology of microorganisms; 11:th edition, Pearson Education Inc. 2006; ISBN 0-13-196893-9, pages 335-337 and 909-912
3. Handbok, illustra puReTaq ready-to-go PCR Beads, GE Healthcare 2006, pages 10, 15
4. Cindy H. Nakatsu, Krassimira Hristova, Satoshi Hanada, Xian-Ying Meng, Jessica R. Hanson, Kate M. Scow and Yoichi Kamagata; Methylibium petroleiphilum gen. nov., sp. nov., a novel methyl tert-butyl ether-degrading methylotroph of the
Betaproteobacteria; International Journal of Systematic and Evolutionary Microbiology (IUMS); 2006, vol. 56, pages 983-989
5. Peter F. Dunfield, Valentina N. Khmelenina, Natalia E. Suzina, Yuri A. Trotsenko and Svetlana N. Dedysh; Methylocella silvestris sp. nov., a novel methanotroph isolated from an acidic forest cambisol; International Journal of Systematic and Evolutionary Microbiology (IUMS); 2003, vol. 53, pages 1231-1239
6. Karin Kirstein, Eberhard Bock; Close genetic relationship between Nitrobacter
hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases, Archives of Microbiology; 1993; vol. 160; pages 447-453
Appendix 1
Recipes
LB-media
10 g. tryptone 5 g. yeast extract 5 g. NaCl 18 g. Agar-agar 1 litre distilled water Ampicillin 50 µg/ml pH adjusted to 7,5.TBE-buffer
0,089 M Tris-borat 0, 0025 M EDTA
Reference: Bernard Perbal, A practical guide to molecular cloning, John Wiley & Sons Inc., 1988 USA, ISBN 0-471-85070-5, page 349
Media for pure culture
ATCC-480 NaNO2 212 mg/l CaCl2 1 mg/l MgSO4*7H2O 100 mg/l K2HPO4 2 drops 1,74 % Iron 1 mg/l Trace elements: MnCl2*4H2O 100 µg/l CuSO4 10 µg/l Na2MoO4*2H2O 50 µg/l CoCl2*6H2O 1 µg/l ZnSO4*7H2O 50 µg/lAppendix 2
Results of different PCR-programmes
Figure 1. A PCR-programme, from a previous study of the diversity of Nitrobacter in soil samples, tested on sludge samples. The figure shows: Well 1) 100 bp size marker 2) Negative control 3) Positive control 4-7) DNA-extracted sludge samples
Figure 2. PCR-products that were obtained when the optimized December programme was applied on the February samples. The figure shows: Well 1)100 bp size marker 2) Negative control 3) Positive control 4-5) DNA-extracted sludge samples dec-08 6-9) DNA-extracted sludge samples feb-09
