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View all other units

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Head of the Chair: prof. Maia Kivisaar
Contact details: Riia 23-104, Phone: +372 737 5036, email: maia.kivisaar [ät] ut.ee

Research group in microbial genetics

Head of the group:
Professor Maia Kivisaar

Contact: Riia 23-104, Phone +372 7375036, email: maia.kivisaar [ät] ut.ee

Research areas:

Molecular mechanisms of evolution of bacteria under stressful conditions

Identification of new genes affecting mutation frequency and elucidation of their role in a network affecting mutational processes

Usage of bacteria in environmentally friendly technologies

Research description

People

Heili Ilves, researcher, PhD, heili.ilves [ät] ut.ee
Signe Saumaa, researcher, PhD, signe.saumaa [ät] ut.ee
Signe Viggor, researcher, PhD, signe.viggor [ät] ut.ee
Merike Jõesaar, researcher, PhD, merike.joesaar [ät] ut.ee
Anne Menert, researcher, PhD, anne.menert [ät] ut.ee
Tatjana Jatsenko, doctoral student, tanjaja [ät] ut.ee
Katren Mikkel, doctoral student, katren.mikkel [ät] ut.ee
Tanel Ilmjärv, doctoral student, tanel.ilmjarv [ät] ut.ee
Kärt Ukkivi, doctoral student, kart.ukkivi [ät] ut.ee
Mari Tagel, doctoral student, mari.tagel [ät] ut.ee
Triin Korb, doctoral student, triin.korb [ät] ut.ee
Ingrem Metsik, doctoral student, ingrem [ät] windowslive.com
Karl Jürgenstein, master student
Liselle Luks, master student
Kadri Kurg, master student
Lauri Leas, bachelor student
Lea Lopp, bachelor student
Jaroslav Panjukov, bachelor student

Research

In nature, bacteria are constantly confronted with variable and stressful environments. Under such conditions bacteria grow very slowly if at all, but despite the reduced amount of DNA replication mutants arise that are able to take over bacterial populations. This process is known as stationary-phase mutagenesis. Mechanisms of stationary-phase mutagenesis are important in generation of resistance to antibiotics, host invasion by pathogenic bacteria, and evolution of new catabolic pathways for degradation of xenobiotic compounds and evolving bacteria-plant interactions. The genus Pseudomonas represents one of the prominent groups of bacteria including both pathogenic and non-pathogenic species. The main goal of our research is to study molecular mechanisms of genetic adaptation of bacteria under conditions of environmental stress by using P. putida and P. aeruginosa as model organisms.

Molecular mechanisms of evolution of bacteria under stressful conditions

We have previously shown that the spectrum of mutations observed in stationary-phase populations of P. putida differs from that occurring among mutants of actively growing bacteria. We have observed that enzymes participating in nucleotide excision repair NER play an important role in generation of mutations in stationary-phase bacteria. Possibly, NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic. In addition to replicative DNA polymerase Pol III and DNA polymerase I, specialized DNA polymerases Pol II, Pol IV and Pol III homologue DnaE2 (ImuC) are found in pseudomonads. Pol IV-dependent mutagenesis causes a remarkable increase in the frequency of accumulation of 1-bp deletion mutants in P. putida populations starved for more than one week. We have also observed that both ImuC and Pol IV (DinB) can confer a protective role against DNA alkylation damage in pseudomonads, whereas mutagenesis induced by DNA alkylation damage is largely dependent on ImuC. Our current research is connected with further elucidation of molecular mechanisms of mutagenic processes and evolution of bacteria exposed to environmental stress by focusing on the role of specialized (potentially error-prone) DNA polymerases in DNA repair synthesis. For example, we are investigating whether specialized DNA polymerases could participate in NER-dependent mutagenesis and have interconnections with other DNA repair systems.

Compared to adaptation of pathogenic bacteria, mutational processes operating in natural populations of bacteria living in polluted environment are still unexplored. Our results indicate that a large proportion of pollutants-degrading indigenous bacteria exhibit a potential for induced mutagenesis under the conditions of DNA damage due to the presence of error-prone DNA polymerases. Our studies will address contribution of these DNA polymerases to evolvability of indigenous bacteria in the presence of environmental pollutants. In addition, to understand adaptation mechanisms of pollutants-degrading bacterial populations at molecular level, laboratory evolution experiments with bacteria, coupled with classical genetics and modern DNA sequence analysis and resequencing technologies will be performed.

Identification of new genes affecting mutation frequency and elucidation of their role in a network affecting mutational processes

We are also interested in identification of a wider network underlying the mutagenesis and evolvability of microbial populations under environmental stress. By combining a newly constructed papillation assay developed in our laboratory and transposon mutagenesis we have already identified and verified several novel genes (e.g., truA, gacS, mpl) affecting mutation frequency in P. putida. This assay and other similar assay systems developed by us can be used in further studies of mutation frequency-affecting genes by performing transposon mutants’ library screenings in strains lacking certain DNA repair enzymes or DNA polymerases, and in the presence of sublethal doses of antibiotics, aromatic pollutants and nano-particles. Functional studies of the identified genes are currently in progress to elucidate their performance in the network affecting mutagenic processes in bacteria.

Usage of bacteria in environmentally friendly technologies

In addition to the basic research we are collaborating with other research groups in order to work out solutions based on microbial processes which would allow removal of environmental pollution and facilitate bioleaching of metals from organometallic complexes-containing ores.

(1) Our earlier studies have been connected with elucidation of mechanisms of regulation of phenol degradation genes pheBA and other catabolic genes involved in pollutants degradation. This competence is used in the ERANET Inno Indigo project “Wastewater reuse: improving the odds by understanding natural attenuation“, where partners from India, Portugal and Estonia are participating. The project aims to analyse the natural attenuation capacity at a wastewater treatment plant and work out ways for improving the degradation process by bioaugmentation. The strains isolated from Surge Pond Sludge and Clarifier samples, which were obtained during our visit to Indian partner’s Institute and wastewater treating plant at Mumbai (URAN), are examined for the ability to use different aromatic and aliphatic compounds and for the presence for corresponding catabolic genes. Bacterial strains with best stress tolerance and biodegradative capacity will be selected for bioaugmentation of crude oil waste degradation.

(2) We are participating in RITA1/01-01 subproject „Bioleaching of metals from Estonian graptolite argillite“. This subproject is a part of the larger interdisciplinary project aiming to provide information on the best available and new innovative technologies for utilization of existing mineral resources and information on properties and possible processing technologies for potential resources in Estonia. Consortium of scientists of different specialties from University of Tartu, Tallinn Technical University, and Geological Survey of Estonia participate in this project. Graptolite argillite (GA) is a potential source for V, Mo, Ni, U, Re and other elements. Metals are in GA as sulfides or in the composition of organometallic compounds. While the role of pyrite oxidation microorganisms in bioleaching of metals is well-studied, little is known about the microbial decomposition of organometallic complexes. Microbial degradation of organometallic complexes and bioleaching of metals would allow valorization of argillite as an environmentally harmful byproduct accompanying mining of other ores. Our research is focused on development and optimisation of consortia of indigenous microorganisms by using those isolated by us previously and by taking new samples of indigenous microorganisms found in the locality of deposit. Medium and conditions for biodegradation of the organic matter of GA and bioleaching of metals will be elaborated.

We are also collaborating with Centre of Synthetic Biology established by Institute of Technology, University of Tartu (participation in ASTRA infrastructure proposal and in Teaming proposal). Soil bacterium Pseudomonas putida could potentially be used as cell factories in various biotechnological settings because of its good genetic accessibility, metabolic versatility and high tolerance to toxic and harsh conditions met in industrial processes. Our competence can be used for genetic manipulation of this organism, monitoring stability and stress levels of engineered strains and further optimization of cell factories by adaptive evolution.

Publications

Selected publications:

1. Ilmjärv, T., Naanuri, E., Kivisaar, M. (2017). Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria. PLoS ONE, e0182484, doi:10.1371/journal.pone.0182484

2. Sidorenko, J., Jatsenko, T., Kivisaar, M. (2017). Ongoing evolution of Pseudomonas aeruginosa PAO1 sublines complicates studies of DNA damage repair and tolerance. Mutat. Res. 797-799:26-37. doi:10.1016/j.mrfmmm.2017.03.005

3. Jatsenko, T., Sidorenko, J., Saumaa, S., Kivisaar, M. (2017). DNA polymerases ImuC and DinB are involved in DNA alkylation damage tolerance in Pseudomonas aeruginosa and Pseudomonas putida. PLoS ONE, e0170719, doi: 10.1371/journal.pone.0170719

4. Tagel, M., Tavita, K., Hõrak, R., Kivisaar, M., Ilves, H. (2016) A novel papillation assay for the identification of genes affecting mutation rate in Pseudomonas putida and other pseudomonads. Mutat.Res. 790:41-55. doi: 10.1016/j.mrfmmm.2016.06.002.

5. Paris, Ü., Mikkel K, Tavita K, Saumaa S, Teras R, Kivisaar M. (2015) NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida. DNA Repair 31:11-8. doi: 10.1016/j.dnarep.2015.04.005.

6. Sidorenko, J., Ukkivi, K., and Kivisaar, M. (2015) NER enzymes maintain genome integrity and suppress homologous recombination in the absence of exogenously induced DNA damage in Pseudomonas putida. DNA Repair 25:15-26. doi: 10.1016/j.dnarep.2014.11.001.

7. Martínez-García, E., Jatsenko, T., Kivisaar, M., and de Lorenzo, V. (2015) Freeing Pseudomonas putida KT2440 of its proviral load strengthens endurance to environmental stresses. Environ Microbiol. 17:76-90. doi: 10.1111/1462-2920.12492.

8. Mielecki, D., Saumaa, S., Wrzesiński, M., Maciejewska, A.M., Żuchniewicz, K., Sikora, A., Piwowarski, J., Nieminuszczy, J., Kivisaar, M., Grzesiuk, E. (2013) Pseudomonas putida proteins AlkA and AkB comprise different defense systems for the repair of alkylation damage to DNA – in vivo, in vitro and in silico studies. PLoS One. 8:e76198. doi: 10.1371/journal.pone.0076198.

9. Juurik, T., Ilves, H., Teras, R., Ilmjärv, T., Tavita, K., Ukkivi, K., Teppo, A., Mikkel, K., and Kivisaar, M. (2012) Mutation frequency and spectrum of mutations vary at different chromosomal positions of Pseudomonas putida. PLOS One 7:e48511. doi: 10.1371/journal.pone.0048511.

10. Tavita, K., Mikkel, K., Tark-Dame M., Jerabek, H., Teras, R., Sidorenko J., Tegova, R., Tover, A., Dame, R.T., and Kivisaar, M. (2012) Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target. Mutat. Res. 737:12-24.

11. Kivisaar, M. (2011). Evolution of catabolic pathways and their regulatory systems in synthetic nitroaromatic compounds degrading bacteria. Mol. Microbiol. 82:265-268.

12. Sidorenko, J., Jatsenko, T., Saumaa S., Teras, R., Tark-Dame, M., Hõrak R., and Kivisaar, M. (2011). Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida. Mutat. Res. 717(1-2):63-77.

13. Kivisaar, M. (2010). Mechanisms of stationary-phase mutagenesis in bacteria: mutational processes in pseudomonads. FEMS Microbiol. Lett. 312:1-14.

14. Jatsenko, T., Tover, A., Tegova, R., and Kivisaar, M. (2010) Molecular characterization of Rif^r mutations in Pseudomonas aeruginosa and Pseudomonas putida. Mutat. Res. 683:106-114.

15. Kivisaar, M. (2009). Degradation of nitroaromatic compounds: a model to study evolution of metabolic pathways. Mol. Microbiol. 74:777-781.

16. Tarassova, K., Tegova, R., Tover, A., Teras, R., Tark, M., Saumaa, S., and Kivisaar, M. (2009) Elevated mutation frequency in survival population of carbon-starved rpoS-deficient Pseudomonas putida is caused by reduced expression of superoxide dismutase and catalase. J. Bacteriol. 191:3604-3614.

17. Teras, R., Jakovleva, J., and Kivisaar, M. (2009) Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652. Microbiology 155:1203-1214.

18. Tark, M., Tover, A., Koorits, L., Tegova, R., and Kivisaar, M. (2008) Dual role of NER in mutagenesis in Pseudomonas putida. DNA Repair 7:20-30.

19. Saumaa, S., Tover, A., Tark, M., Tegova, R., and Kivisaar M. (2007) Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida. J. Bacteriol. 189:5504-5514.

20. Putrinš, M., Tover, A., Tegova, R., Saks, Ü., and Kivisaar M. (2007) Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida. Microbiology 153:1860-1871.

21. Koorits, L., Tegova, R., Tark, M., Tarassova, K., Tover, A., and Kivisaar M. (2007) Study of involvement of ImuB and DnaE2 in stationary-phase mutagenesis in Pseudomonas putida. DNA Repair 6:863-868.

22. Kivistik, P.A., Putrinš, M., Püvi, K., Ilves, H., Kivisaar, M., and R. Hõrak. (2006) ColRS two-component system regulates membrane functions and protects Pseudomonas putida against phenol. J. Bacteriol. 188:8109-8117.

23. Saumaa, S., Tarassova, K., Tark, M., Tover, A., Tegova, R., and Kivisaar M. (2006) Involvement of DNA mismatch repair in stationary-phase mutagenesis during prolonged starvation of Pseudomonas putida. DNA Repair 5:505-514.

24. Tark, M., A. Tover, K. Tarassova, R. Tegova, G. Kivi, R. Hõrak, and Kivisaar M. (2005) A DNA polymerase V homologue encoded by TOL plasmid pWW0 confers evolutionary fitness on Pseudomonas putida under conditions of environmental stress. J. Bacteriol. 187:5203-5213.

25. Tegova, R., Tover, A., Tarassova, K., Tark, M., and Kivisaar, M. (2004) Involvement of error-prone DNA polymerase pol IV on stationary phase mutagenesis in Pseudomonas putida. J. Bacteriol. 186:2735-44.

26. Neumann, G., Teras, R., Monson, L., Kivisaar, M., Schauer, F., and Heipieper, H.J. (2004) Simultaneous degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and adaptation. Appl. Environ. Microbiol. 70:1907-1912.

27. Ilves, H., Hõrak, R., Teras, R., and Kivisaar, M. (2004) IHF is limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase. Mol. Microbiol 51:1773-85.

28. Hõrak, R., Ilves, H., Pruunsild, P., Kuljus, M., and Kivisaar, M. (2004) The ColR-ColS two-component signal transduction system is involved in regulation of Tn4652 transposition in Pseudomonas putida under starvation conditions. Mol. Microbiol. 54:795-807.

29. Kivisaar, M. (2003) Stationary phase mutagenesis: mechanisms that accelerate adaptation of microbial populations under environmental stress. Environ. Microbiol. 5: 814-827.

30. Saumaa, S., Tover, A., Kasak, L., and Kivisaar, M. (2002) Different spectra of stationary-phase mutations in early-arising versus late-arising mutants of Pseudomonas putida: involvement of the DNA repair enzyme MutY and the stationary-phase sigma factor RpoS. J. Bacteriol. 184:6957-6965.

31. Ilves, H., Hõrak, R., and Kivisaar, M. (2001) Involvement of sigma(S) in starvation-induced transposition of Pseudomonas putida transposon Tn4652. J. Bacteriol. 183:5445-5448.

32. Tover, A., Ojangu, E.L., and Kivisaar, M. (2001) Growth medium composition-determined regulatory mechanisms are superimposed on CatR-mediated transcription from the pheBA and catBCA promoters in Pseudomonas putida. Microbiology 147:2149-2156.

33. Ojangu, E., Tover, A., Teras, R., and Kivisaar, M. (2000) Effect of combination of different –10 hexamers and downstream sequences on stationary phase-specific sigma factor s^S-dependent transcription in Pseudomonas putida. J. Bacteriol. 182:6707-6713

34. Teras, R., R. Hõrak, and Kivisaar, M. (2000) Transcription from fusion promoters generated during transposition of transposon Tn4652 is positively affected by integration host factor in Pseudomonas putida. J. Bacteriol.182:589-598.

35. Tover, A., Zernant, J., Chugani, S.A., Chakrabarty, A.M., and Kivisaar, M. (2000) Critical nucleotides in the interaction of CatR with the pheBA promoter: conservation of the CatR-mediated regulation mechanisms between the pheBA and catBCA operons. Microbiology. 146: 173-183.

36. Hõrak, R., and Kivisaar, M. (1999) Regulation of transposase of Tn4652 by the transposon-encoded protein TnpC. J. Bacteriol. 181: 6312-6318.

37. Kallastu, A., Hõrak, R., and Kivisaar, M. (1998) Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida. J. Bacteriol. 180:5306-5312.

38. Hõrak, R., and Kivisaar, M. (1998) Expression of the transposase gene tnpA of Tn4652 is positively affected by integration host factor. J. Bacteriol. 180:2822-2829.

39. Kasak, L., Hõrak, R., and Kivisaar, M. (1997) Promoter-creating mutations in Pseudomonas putida: a model system for the study of mutation in starving bacteria. Proc. Natl. Acad. Sci. U.S.A. 94:3134-3139.

40. Parsek., M.,R., Kivisaar, M., and Chakrabarty, A., M. (1995) Differential DNA bending induced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters. Mol. Microbiol. 15:819-828.

41. Kasak, L., Hõrak, R., Nurk, A., Talvik, K., and Kivisaar, M. (1993) Regulation of the catechol 1,2-dioxygenase and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85. J. Bacteriol. 175:8038-8042.

42. Nurk, A., Tamm, A., Hõrak, R., and Kivisaar, M. (1993) In vivo generated fusion promoters in Pseudomonas putida. Gene 127:23-29.

43. Nurk, A., Kasak, L., and Kivisaar, M. (1991) Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida. Gene 102:13-18.

44. Kivisaar, M., Kasak, L., and Nurk, A. (1991) Sequence of the plasmid-encoded catechol 1,2-dioxygenase-expressing gene, pheB, of phenol-degrading Pseudomonas sp. strain EST1001. Gene 98:15-20.

45. Kivisaar, M., Hõrak, R., Kasak, L., Heinaru, A., and Habicht, J. (1990) Selection of independent plasmids determining phenol degadation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase. Plasmid 24:25-36.

46. Kivisaar, M., Habicht, J., and Heinaru, A. (1989) Degradation of phenol and m-toluate in Pseudomonas sp. strain EST1001 and its transconjugants is determined by a multiplasmid system. J. Bacteriol. 171:5111-5116.

Patent application

Menert, A.; Kivisaar, M.; Sipp Kulli, S.; Heinaru, A.; Maidre, T. Method for decomposition of the metallorganic matter of graptolite-argillite by microbial consortium; Owner: BiotaTec OÜ; Authors: Priority number: WO/2017/140324; Priority date: 16.02.2016; Published: 24.08.2017.

Bacterial stress tolerance research group

Head of the group:
Senior Research Fellow Rita Hõrak

Contact: Riia 23-106, Phone +372 7374077,
email: rita.horak [ät] ut.ee

Research areas:

Signalling pathways for stress tolerance

Fitness effects of toxin-antitoxin systems

Research description

People

Rita Hõrak, senior research fellow, PhD, rita.horak [ät] ut.ee
Andres Ainelo, PhD student, MSc, andres.ainelo [ät] ut.ee
Kadi Ainsaar, PhD student, MSc, kadi.ainsaar [ät] ut.ee
Sirli Luup, PhD student, MSc, sirli.luup [ät] ut.ee

Research

ColRS – signal transduction pathway for metal tolerance

Successful survival of bacteria in ever-changing conditions depends on their ability to monitor the environment and to translate external signals into adaptive responses, mostly by adjusting their gene expression according to new situations. Two-component systems consisting of a transmembrane sensor kinase and a cytoplasmic response regulator are important signal pathways in bacteria. The ColRS signaling pathway senses the excess of zinc, iron, manganese and cadmium and contributes to metal tolerance of Pseudomonas putida. We aim to define the regulatory network operating downstream of the ColRS signaling to enlighten the molecular mechanisms of metal tolerance of P. putida. We also analyze the possible interconnections between ColRS signaling and the three-component TonB system, which is important for the energy transduction between outer and inner membrane.

Fitness effects of toxin-antitoxin systems in Pseudomonas putida

Toxin-antitoxin (TA) systems code for two proteins: one is toxic to vital cellular processes and the other functions as an antidote of the toxin. Bacterial genomes contain many copies of these potentially poisonous gene pairs. It has been proposed that TA systems contribute to stress tolerance, as they are able to shift the cells to a dormant state. However, as studies conducted so far have resulted in controversial outcomes, the biological importance of TA systems still remains unclear. We aim to evaluate the costs and benefits of genomic TA pairs in the biology of P. putida. We are performing a systematic screening of 15 predicted genomic TA systems by examining the consequences of antitoxin deletions as well as the fitness effects of removal of all 15 TA operons from the P. putida genome. Our studies will hopefully reveal whether TA operons are just selfish DNA elements or impact bacterial fitness and stress survival.

Publications

Selected publications:

1. Hõrak, R. and H. Tamman. 2017. Desperate times call for desperate measures: benefits and costs of toxin-antitoxin systems. Curr Genet. 63(1):69-74.

2. Talavera, A., H. Tamman, A. Ainelo, S. Hadži, A. Garcia-Pino, R. Hõrak, A. Konijnenberg, and R. Loris. 2017. Production, biophysical characterization and crystallization of Pseudomonas putida GraA and its complexes with GraT and the graTA operator. Acta Crystallogr F Struct Biol Commun. 73(Pt 8):455-462.

3. Tamman, H., A. Ainelo, M. Tagel, and R. Hõrak. 2016. Stability of the GraA antitoxin depends on the growth phase, ATP level, and global regulator MexT. J Bacteriol. 198(5):787-796.

4. Ainelo, A., H. Tamman, M. Leppik, J. Remme, and R. Hõrak. 2016. The toxin GraT inhibits ribosome biogenesis. Mol Microbiol. 100(4):719-734.

5. Mumm, K., K. Ainsaar, S. Kasvandik, T. Tenson, and R. Hõrak. 2016. Responses of Pseudomonas putida to zinc excess determined at the proteome level: pathways dependent and independent of ColRS. J Proteome Res. 15(12):4349-4368.

6. Tamman, H., A. Ainelo, K. Ainsaar, and R. Hõrak. 2014. A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida. J Bacteriol. 196(1):157-69.

7. Ainsaar, K., K. Mumm, H. Ilves, and R. Hõrak. 2014. The ColRS signal transduction system responds to the excess of external zinc, iron, manganese, and cadmium. BMC Microbiol. 14:162.

8. Putrinš, M., A. Ainelo, H. Ilves, and R. Hõrak. 2011. The ColRS system is essential for the hunger response of glucose-growing Pseudomonas putida. BMC Microbiol. 11:170.

9. Putrinš, M., H. Ilves, L. Lilje, M. Kivisaar, and R. Hõrak. 2010. The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria. BMC Microbiol. 10:110.

10. Putrinš, M., H. Ilves, M. Kivisaar, and R. Hõrak. 2008. ColRS two-component system prevents lysis of subpopulation of glucose-grown Pseudomonas putida. Environ Microbiol. 10(10):2886-2893.

11. Kivistik, P.A., M. Putrinš, K. Püvi, H. Ilves, M. Kivisaar, and R. Hõrak. 2006. The ColRS two-component system regulates membrane functions and protects Pseudomonas putida against phenol. J Bacteriol. 188(23):8109-17.

Research group in bacterial lifestyles

Head of the group:
Associate Professor Riho Teras

Contact: Riia 23, Tartu
Phone: +372 737 6038
email: riho.teras [ät] ut.ee

Research areas:

Factors of biofilm development

Global regulators of bacteria

Topics:

Biofilm of Pseudomonas putida: attachment, switching from planktonic lifestyle to sessile, involvement of global regulators in biofilm formation, P. putida’s adhesins LapA and LapF, extracellular factors, the hydrophobicity of bacterial surface, colonization of plant roots

Global regulators of P. putida: global regulator Fis, GacS/GacA signal system, the involvement of ROS in colonization of plant roots

Research description

People

Riho Teras, associate professor, Ph.D., riho.teras [ät] ut.ee
Annika Teppo, doctoral student, MSc, annika.teppo [ät] ut.ee
Marge Puhm, master student
Kadri Samuel, bachelor student
Johanna Hendrikson, bachelor student

Research

Biofilm is an ancient lifestyle of bacteria – the first structures of this kind of bacterial life appeared billions of years ago. The functionality and structure of a biofilm is often compared to a city – it has roads for transport and skyscrapers for living in. Indeed, like a city, every biofilm has its own structure and has had its unique development, depending on the bacterial species and environment. Biofilms have been having a remarkable effect on the life on Earth since appearance, and now, in modern human society, the negative impact of biofilm causes approximately 6 billions of dollars of economic loss every year.

We are interested in regulators and factors that enhance biofilm formation in Pseudomonas putida, a cosmopolitan bacterium that often colonizes plant roots and has a positive impact on plant growth. Thereby P. putida is a significant bacterium for agricultural applications. To defend the plant from pathogenic microorganisms, P. putida has to attach to roots and colonize the roots surface, including the root tips which grow up to 10 mm per day. We have shown that the colonization of barley root tips depends on biofilm formation and a global regulator Fis. The overexpression of fis has a negative impact on migration to the root tips due to enhanced biofilm formation, the sessile lifestyle of bacteria. As plant roots secrete reactive oxygen species (ROS), we also examine the effects of ROS on the colonization ability of P. putida.

The global regulator Fis binds and bends DNA, and thereby, its functionality appears in processes that need alteration of DNA topology, including transposition and regulation of transcription. However, our prime aim was to ascertain Fis-regulated genes that are involved in biofilm formation of P. putida. Genes of adhesins lapA and lapF are Fis-regulated and have a significant role in P. putida biofilm formation. We identified six sigma70-type promoters for the transcription of lapA and two Fis-binding sites for the activation of lapA expression. These results add Fis to the list of lapA transcriptional regulators already including the GacS/GacA signal system, FleQ, (p)ppGpp, c-di-GMP; and point to a sophisticated transcriptional regulation. Contrary to the complex transcriptional regulation of lapA, for lapF, we identified only one promoter and one Fis-binding site, which overlapped with the promoter. We continue the study to ascertain the network regulating the transcription of lapA.

The study for the function of LapF protein has been most intriguing. Similarly to LapA, LapF is a surface protein, but unlike LapA it is expressed mostly in the stationary phase or in the mature biofilm. We showed that lapF-deletion decreased cell surface hydrophobicity and the presence of LapF passively influences P. putida’s sensitivity to toxic hydrophobic and hydrophilic compounds. Hydrophobicity of bacterial surface has been described as an essential factor for cell-cell contact in the mature biofilm and cell aggregation. However, the lapF-deletion does not affect mature biofilm in LB medium. Therefore we are searching for a potential function for LapF.

Confocal micrograph of aggregated P. putida strain F15 in semisolid LB medium. Cells are stained with membrane-selective dye FM-142.

Confocal micrograph of ROS-producing P. putida strain F15. Cells are stained with ROS-sensitive dye rhodamin-123.

Publications

Teppo A, Lahesaare A, Ainelo H, Samuel K, Kivisaar M, et al. (2018) Colonization efficiency of Pseudomonas putida is influenced by Fis-controlled transcription of nuoA-N operon. PLoS One 13: e0201841

Ainelo, H., A. Lahesaare, A. Teppo, M. Kivisaar & R. Teras, (2017) The promoter region of lapA and its transcriptional regulation by Fis in Pseudomonas putida. PloS one 12: e0185482.

Lahesaare, A., H. Ainelo, A. Teppo, M. Kivisaar, H.J. Heipieper & R. Teras, (2016) LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida. PloS one 11: e0166078.

Lahesaare, A., H. Moor, M. Kivisaar & R. Teras, (2014) Pseudomonas putida Fis binds to the lapF promoter in vitro and represses the expression of LapF. PloS one 9: e115901.

Moor, H., A. Teppo, A. Lahesaare, M. Kivisaar & R. Teras, (2014) Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF. Microbiology (Reading, England) 160: 2681-2693.

Research group in microbial ecology

Head of the group:
Professor Ain Heinaru

Contact: Riia 23-101, Tartu
Phone: +372 737 5012
email: ain.heinaru [ät] ut.ee

Research areas:

Structure, functioning and redundancy of biodegradative pathways and plasmids, evolution of new catabolic features in environmental bacteria.
Microbial ecology of the Baltic Sea and adjoining polluted oil shale industry areas.
Bioaugmentation performances for cleaning up polluted water and sediments.
Collection of environmental and laboratory microbial strains (CELMS).

Research description

People

Ain Heinaru, Professor, PhD
ain.heinaru [ät] ut.ee

Eve Naanuri, Senior Research Fellow/

Specialist, PhD, eve.naanuri [ät] ut.ee

Eeva Heinaru, Research Fellow/

Project Manager, MSci, eeva.heinaru [ät] ut.ee

Merike Jõesaar, Research Fellow, PhD

merike.joesaar [ät] ut.ee

Signe Viggor, Research Fellow, PhD

signe.viggor [ät] ut.ee

Jaanis Juhanson, PhD, Post-Doctoral Student (Sweden)

Jekaterina Jutkina, PhD, Post-Doctoral Student (Sweden)

Research

Nature contains chemical compounds that are degraded by the catabolic activities of microorganisms to generate the energy and to support the world-wide life cycle. Some of the compounds (aromatic and aliphatic hydrocarbons, heavy metals etc.) are highly toxic and mutagenic for organisms. However, several bacteria are able to degrade these compounds. By using natural selection and the molecular genetic engineering methods it is possible to generate supermicrobes applicable for cleaning up of polluted areas.

Structure, functioning and redundancy of biodegradative pathways and plasmids, evolution of new catabolic features in environmental bacteria

Bacterial catabolic genes are located on extrachromosomal plasmids and/or in chromosomes. Our special focus is on the structure and functioning of these genes and operons. We perform nucleotide sequencing of whole genomes of bacteria in order to understand how these catabolic structures have evolved. We are also interested in the functional redundancy of catabolic modules to verify the best catabolic activities both in indigenous and laboratory constructed bacterial strains. Essential part of this work is the determination of enzyme activities and identification of intermediates of the catabolic pathways and studying the role of these intermediates in the performance of catabolic functions.

Microbial ecology of the Baltic Sea and adjoining polluted oil shale industry areas

The Baltic Sea is unique among the seas of the world, characterized with the busiest maritime traffic, a high population along its coast area, and the specific unique ecosystem. Large rivers from highly industrialized and agriculturally intensive countries bring high loads of agricultural nutrients (like nitrogen and phosphorous) and hazardous compounds (herbicides, antibacterial agents, oil products, phenolic chemicals etc.) into the Baltic Sea. Therefore our latest research topic has been the elucidation of the biodegradative potential of microbial communities in the Baltic Sea sediment and surface water samples. Culture dependent and independent methods have been used to provide a more accurate picture about the complex microbial communities. Some of the isolated bacterial strains have extremely interesting characteristics useful for the practical purposes in degradation of crude oil derived petroleum hydrocarbons. That is why in these cases we are generating special research projects based on particular strains. This work is done in collaboration with research institutions from Finland and Russia. Another project is ongoing with researchers from Portugal and India to analyze the natural attenuation capacity of the wastewater treatment plant of crude oil refinery and find ways for improving the degradation process. This project targets a global priority issue, i.e., reuse of industrial wastewater.

Bioaugmentation performances for cleaning up polluted water and sediments

We have long time successful experiences at bioaugmentation of polluted water and sediments. In case of introducing biomass of laboratory selected bacteria to the open environment the key molecular markers (DNA fingerprints) are determined in sense to follow what happens with those bacteria in open nature. Before the field bioaugmentation experiments the behavior of bacteria in laboratory in microcosm experiments mimicking real pollution conditions is always studied.

Collection of the environmental and laboratory microbial strains (CELMS)

We are responsible for the Estonian National collection of non-medical environmental and laboratory microbial strains (CELMS, http://eemb.ut.ee/ ). Collection contains great variety of indigenous environmental bacteria characterized by DNA sequences of important catabolic regions, species determining gene sequences. Part of the strains has been subjected to whole genome sequence determination. This is a basic facility for our laboratory research. The new bacterial strains obtained within research projects conducted in our department are stored at the culture collection. The intellectual property agreement between author of the bacterial strain and official representative of collection guarantees author’s rights.

Publications

Selected publications

Yan, L.; Yu, D.; Hui, N.; Naanuri, E.; Viggor, S.; Gaforov, A.; Sokolov, S.; Heinaru, A.; Romantschuk, M. (2018). Distribution of archaeal communities along the coast of the Gulf of Finland and their response to oil contamination. Frontiers in Microbiology, 9, 1−19.
Jõesaar, Merike; Viggor, Signe; Heinaru, Eeva; Naanuri, Eve; Mehike, Maris; Leito, Ivo; Heinaru, Ain (2017). Strategy of Pseudomonas pseudoalcaligenes C70 for effective degradation of phenol and salicylate. PLoS ONE, 12 (3), 1−17.
Heinaru, Eeva; Naanuri, Eve; Grünbach, Maarja; Jõesaar, Merike; Heinaru, Ain (2016). Functional redundancy in phenol and toluene degradation in Pseudomonas stutzeri strains isolated from the Baltic Sea. Gene, 589, 90−98.
Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain. (2015). Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water. Marine Pollution Bulletin, 101 (2), 507−516.
Tiirik, Kertu; Nõlvak, Hiie; Oopkaup, Kristjan; Truu, Marika; Preem, Jens-Konrad; Heinaru, Ain; Truu, Jaak (2014). Characterization of the bacterioplankton community and its antibiotic resistance genes in the Baltic Sea. Biotechnology and Applied Biochemistry, 61 (1), 23−32.
Vedler, Eve; Heinaru, Eeva; Jutkina, Jekaterina; Viggor, Signe; Kõressaar, Triinu; Remm, Maido; Heinaru, Ain (2013). Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterized by comparative analysis of catabolic genes. Systematic and Applied Microbiology, 36 (8), 525−532.
Jutkina, Jekaterina; Hansen, Lars; Li, Lili; Heinaru, Eeva; Vedler, Eve; Jõesaar, Merike; Heinaru, Ain (2013). Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids. Plasmid, 70 (3), 393−405.
Viggor, Signe; Juhanson, Jaanis; Jõesaar, Merike; Mitt, Mario; Truu, Jaak; Vedler, Eve; Heinaru, Ain (2013). Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil, shale oil or diesel fuel. Microbiological Research, 168 (7), 415−427.
Jutkina, Jekaterina; Heinaru, Eeva; Vedler, Eve; Juhanson, Jaanis; Heinaru, Ain (2011). Occurrence of plasmids in the aromatic degrading bacterioplankton of the Baltic Sea. Genes, 2 (4), 853−868.
Jõesaar, M.; Heinaru, E.; Viggor, S.; Vedler, E.; Heinaru, A. (2010). Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-cresol-degradative strains of Pseudomonas fluorescens. FEMS Microbiology Ecology, 72 (3), 464−475.

Research group for the study of bacterial and yeast proteins

Head of the group:
Associate Professor Tiina Alamäe

Contact: Riia 23-302, Tartu
Phone: +372 737 5013
email: tiina.alamae [ät] ut.ee

Research areas:

Bacterial levansucrases and endo-levanases in synthesis of prebiotic sugars

Alpha-glucosidases of yeasts of different phylogenetic age

Research description

People

Tiina Alamäe, associated professor, PhD, tiina.alamae [ät] ut.ee
Triinu Visnapuu, research scientist, PhD, triinu.visnapuu [ät] ut.ee
Karin Ernits, PhD student, MSc, karin.ernits [ät] ut.ee
Katrin Viigand, PhD student, MSc, katrin66 [ät] ut.ee
Kristina Põšnograjeva, MSc student, kristina.poshnograjeva [ät] gmail.com
Aivar Meldre, MSc student, aivarmeldre [ät] gmail.com
Emma-Johanna Sova, undergraduate student, emmajsova [ät] gmail.com

Research

Bacterial levansucrases and endo-levanases as producers of prebiotic poly- and oligosaccharides

Human gut microbiota is currently in the spotlight of science, because it affects human health and well-being. About 1.5 kg of diverse bacteria are inhabiting the human gut thriving mostly at the expense of leftovers from the human table – nondigestible carbohydrates, also named as food fiber. Food fibers act as prebiotics - selective growth promoters of beneficial (probiotic) gut bacteria. Since now, bifodobacteria and lactobacilli are most known probiotic bacteria in the human gut. These bacteria are stimulated for example by inulin (a β 2,1 linked fructose polymer) and galactooligosaccharides – most popular and used prebiotics. As knowledge on gut microbiota composition and roles grows rapidly, food technologists and food scientists are expecting new prebiotics in the market. We study bacterial enzymes that can be used to produce novel prebiotic sugars – levans and levan-type fructooligosaccharides (FOS) from simple table sugar. These products are similar to inulin, but have different linkage (β 2,6) between the fructose residues. To produce levan and FOS, we use extremely stable and active bacterial levansucrase isolated from a plant-associated bacterium Pseudomonas syringae. We have shown that both these preparations stimulate growth of Bacteroides thetaiotaomicron that is an abundant commensal of the human gut. To elaborate medium-scale production of levan-type FOS from bacterial and plant-related levans, we have isolated and charecaterized an endo-levanase of B. thetaiotaomicron. Levan-type FOS serve as substrates for several species of beneficial gut bacteria such as bifidobacteria and lactobacilli, but may also act prebiotically towards novel probiotic bacteria. We are testing the effect of synthesized by us sugar preparations on fecal bacteria in collaboration with groups from Tallinn. As an example of one recent collaborative project on functional food ingredients see (https://www.etis.ee/Projects/projects/Display/f584545f-8fa3-4e5f-9f1d-776d6165cb63). As we are interested in properties of the enzymes we are using, we are also dealing with structure-function studies of levansucrases and the endo-levanase. For the study of mutant and wild-type enzymes, we explore efficient expression systems of proteins in Escherichia coli.

α-glucosidases of yeasts of varied phylogenetic age

α-glucosidases (including maltases and isomaltases) are enzymes that hydrolyze α-glucosidic linkage in various di- and oligosaccharides such as maltose, sucrose, maltotriose, isomaltose and others. These enzymes are required for example in beer-making as the brewing yeast Saccharomyces uses these enzymes to ferment malt sugars into alcohol. Maltases of Saccharomyces yeasts have a quite narrow substrate range: they degrade maltose and maltotriose (both α-1,4 linked), while cannot degrade isomaltose, an α-1,6 linked starch degradation product. For isomaltose degradation, Saccharomyces yeasts have specific enzymes – isomaltases. These two types of specific α-glucosidases most probably evolved from a common promiscuous ancestral protein as proposed in 2012 by Voordeckers and others. We recently showed (Viigand et al. 2016)that resurrected ancMALS and the maltase of a methylotrophic yeast Ogataea (Hansenula) polymorpha are highly similar according to substrate usage and amino acid sequence motifs. As O. polymorpha belongs to an earlier diverged lineage of yeasts, presence of a living “twin” of the hypothetical ancestral promiscuous ancient maltase in O. polymorpha supports the Voordeckers hypothesis. We are currently interested in α-glucosidases of yeasts that belong to different branches of the phylogenetic tree. We will inspect the genomes of these yeasts and search for the genes that may encode α-glucosidases. We will select a set of genes of interest for heterologous overexpression in Escherichia coli. In case of successful expression of a gene, we will study properties of respective enzyme and compare with those of earlier characterized ones. The obtained results will hopefully show some light on phylogenesis of α-glucosidase proteins in pro- and eukaryotes.

Publications

Selected publications

Mardo, Karin; Visnapuu, Triinu; Vija, Heiki; Aasamets, Anneli; Viigand, Katrin; Alamäe, Tiina (2017). A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass. PLOS ONE, 12 (1), e0169989−e0169989
Ernits, K., Viigand, K., Visnapuu, T., Põšnograjeva, K. and Alamäe, T. (2017). Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method. Bio-protocol 7(12): e2349. DOI: 10.21769/BioProtoc.2349.
Bondarenko, Olesja M; Ivask, Angela; Kahru, Anne; Vija, Heiki; Titma, Tiina; Visnapuu, Meeri; Joost, Urmas; Pudova, Ksenia; Adamberg, Signe; Visnapuu, Triinu; Alamäe, Tiina. (2016). Bacterial polysaccharide levan as stabilizing, non-toxic and functional coating material for microelement-nanoparticles. Carbohydrate Polymers, 136: 710−720, 10.1016/j.carbpol.2015.09.093.
Viigand, Katrin; Visnapuu, Triinu; Mardo, Karin; Aasamets Anneli; Alamäe, Tiina (2016). Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to resurrected ancestor protein ancMALS of yeast maltases and isomaltases. Yeast, 33: 415−432, 10.1002/yea.3157.
Adamberg, Kaarel; Tomson, Katrin; Talve, Tiina; Pudova, Ksenia; Puurand, Marju; Visnapuu, Triinu; Alamäe, Tiina; Adamberg, Signe (2015). Levan Enhances Associated Growth of Bacteroides, Escherichia, Streptococcus and Faecalibacterium in Fecal Microbiota. PloS one, 10 (12), e0144042, 10.1371/journal.pone.0144042.
Visnapuu, Triinu; Mardo, Karin; Alamäe, Tiina (2015). Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides. New Biotechnology, 32: 597−605, 10.1016/j.nbt.2015.01.009.
Adamberg, Signe; Tomson, Katrin; Vija, Heiki; Puurand, Marju; Kabanova, Natalja; Visnapuu, Triinu; Jõgi, Eerik; Alamäe, Tiina; Adamberg, Kaarel (2014). Degradation of fructans and production of propionic acid by Bacteroides thetaiotaomicron are enhanced by the shortage of amino acids. Frontiers in Nutrition, 1 (21): 1−10, 10.3389/fnut.2014.00021.
Mardo, Karin; Visnapuu, Triinu; Gromkova, Maria; Aasamets, Anneli; Viigand, Katrin; Vija, Heiki; Alamäe, Tiina. (2014). High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan. Molecules, 19(6): 8434 - 8455.
Mardo, Karin; Visnapuu, Triinu; Vija, Heiki; Elmi, Triin; Alamäe, Tiina (2014). Mutational analysis of conserved regions harbouring catalytic triad residues of the levansucrase protein encoded by lsc-3 (lsc3) gene of Pseudomonas syringae pv. tomato DC3000. Biotechnology and Applied Biochemistry, 61(1): 11 – 22
Suppi, S; Michelson, T; Viigand, K; Alamäe, T. (2013). Repression vs. activation of MOX, FMD, MPP1 and MAL1 promoters by sugars in Hansenula polymorpha: the outcome depends on cell’s ability to phosphorylate sugar . FEMS Yeast Research, 13(2): 219 - 232.
Visnapuu, Triinu; Mardo, Karin; Mosoarca, Cristina; Zamfir, Alina D.; Vigants, Armands; Alamäe, Tiina (2011). Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation. Journal of Biotechnology, 155: 338 - 349.
Visnapuu, T.; Mäe, A.; Alamäe, T. (2008). Hansenula polymorpha maltase gene promoter with sigma 70-like elements is feasible for Escherichia coli-based biotechnological applications: expression of three genomic levansucrase genes of Pseudomonas syringae pv. tomato. Process Biochemistry, 43(4): 414 - 422.
Viigand, K.; Alamae, T. (2007). Further study of the Hansenula polymorpha MAL locus: characterization of the alpha-glucoside permease encoded by the HpMAL2 gene. FEMS Yeast Research, 7(7): 1134 - 1144.
Viigand, K.; Tammus, K.; Alamae, T. (2005). Clustering of MAL genes in Hansenula polymorpha: Cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes. FEMS Yeast Research, 5(11): 1019 - 1028.
Alamae, T.; Parn, P.; Viigand, K.; Karp, H. (2003). Regulation of the Hansenula polymorpha maltase gene promoter in H. polymorpha and Saccharomyces cerevisiae. FEMS Yeast Research, 4(2): 165 - 173.
Liiv, L.; Parn, P.; Alamae, T. (2001). Cloning of maltase gene from a methylotrophic yeast, Hansenula polymorpha. Gene, 265(1-2), 77 - 85.

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Chair of Genetics