Méhi, Orsolya Katinka

English

Méhi Orsolya Katinka

University of Szeged

Volume 55(1):197-212, 2011 Acta Biologica Szegediens is h t t p : / /www.sc i . u - szeged .hu /ABS content , specific ROS scavenging capaci t ies and accl imation potential of leaves under various modera te stress condi t ions . Methods already exist for measur ing total antioxidant capacities and for detect ing enzymes specific to a certain ROS (like peroxidases o r superoxide dismutases) . however some of the ROS are not a imed by specific enzymes and are scavenged by the c o m m o n ef fec t of d i f ferent compounds . The re fo re we developed new methods for measur ing antioxidant capaci t ies of plant samples specif ic to the hydroxy! radical (Maje r et al. 2010b) and the singlet oxygen . (Hideg and Majer 2010) . By correlat ing the ROS specif ic and total ant ioxidant parameters w e showed the impor tance of s tudying the former besides measur ing the widely used latter parameters as well (Ma je r et al. 2010b) . In addi-t ion. w e developed a new method which serves us with an initial screening of photosynthesis in leaves based on digital images (Ma je r et al. 2010a) . Using the above techniques and chlorophyll-fluorescence-based assessment of leaf photochemistry, we studied contributions of preventive and antioxidant processes to high-light tolerance in linden tree (Tilia platyphyllos L.) leaves col lected f r o m sun-exposed and shaded parts of the same tree. Accord ing to our results, l inden sun leaves had 2- t imes s tronger singlet oxygen neutral izing capaci t ies than shade leaves and were able to avoid non-regulated loss of energy under high PAR (Hideg and Majer . 2010) . In sun linden leaves significantly h igher amoun t s of flavonoid glycosides were found and contr ibut ions of various phenol ics to specific ROS capaci t ies are currently investigated. In an other series of exper iments we studied the accl imation potential to UV-B radiation in younge r and older grapevine leaves (Vitis vinifera L. cv. Chardonnay) and found that younger leaves were able to mobi l ize screening p igments and ant ioxidants bet ter and therefore suffered less damage f rom UV-B as compared to o lder ones (Maje r and Hideg 2011) . Hideg É. Majer P (20I0) Factors contributing to the high light tolerance of leaves in vivo - Involvement of photo-protective energy dissipation and singlet oxygen scavenging. Acta Biol Hung 6l(Suppl.): 69-80. Majer P. Sass L . Horváth V G . Hideg É (20i0a) Leaf hue measurements offer a fast, high-throughput initial screening of photosynthesis in leaves. J Plant Physiol 167(I):74-76, Majer P. Stoyanova S, Hideg E (20l()b) Do leaf total antioxidant capacities ( T A C ) reflect specific antioxidant potentials? - A comparison of T A C and reactive oxygen scavenging in tobacco leaf extracts. Journal of Photochem Photobiol В I00( I ):38-43. Majer P. Hideg É ( 2 0 l I ) Are young ones more vigorous? A comparative study of photosynthesis and antioxidant responses of younger and older Vitis leaves under UV-B radiation in a green-house experiment. Abstracts of the 1st Annual Meeting of C O S T Action FA0906 UV4growth; Szeged. Hungary. Supervisors: Éva Hideg. Gábor V. Horváth E-mail: pmajer@brc.hu The role of oxidative stress in antibiotic resistance evolution Orsolya Katinka Mehi E v o l u t i o n a r y Systems B i o l o g y G r o u p , I n s t i t u t e o f B iochemis t ry , B io log i ca l Research Cen t re , H u n g a r i a n A c a d e m y o o f Sciences, Szeged, H u n g a r y T h e emergence of bacteria that are resistant to multiple antibiotics represents an increasingly signif icant threat to public health today. There are a number of mechan i sms whereby bacteria can develop antibiotic resistance including the physical exchange of genet ic material with another organism, the activation of latent mobile genetic e lements ( t ransposons or cryptic genes) and the mutagenesis of its own D N A . Chro-mosomal mutagenesis may arise directly f rom antibiotic-induced oxidative stress, or indirectly, as a consequence of the interaction be tween antibiotic and its target molecu le o r f rom the activity of bac ter ium's er ror-prone D N A polymerases dur ing the repair of D N A lesions. There are at least three major mutational mechanisms which facilitate gradual evolution of bacterial antibiotics resistance. These mecha-nisms include mutators (genotypes with increased, constitutive mutat ion rates). S O S response (a global response that min imizes the lethal and mutagenic consequences of the exposure of cell to DNA d a m a g i n g agents ) and the direct mutagenic effect of reactive oxygen species (ROS) . We investigated how far these three above ment ioned mechan i sms are linked to each other, and what their relative contr ibut ion to ant ibiot ics resistance evolution is. It is known f rom literature that bactericidal (but not bacteriostatic) antibiotics share a c o m m o n lethal pa thway that involves the genera-t ion/accumulat ion of ROS. In our study we focused on the role of ROS in antibiotic resistance evolution. The ef fec t of ROS has a doub le edged sword feature. On one hand ROS production contributes significantly to the killing ef fec t of bactericidal antibiotics. On o ther hand, by directly damaging DNA. ROS accumula t ion increases the muta t ion supply of the bacterial cell which p romotes the appearance of an-tibiotic resistant strains. We employed a series of 10-15 days- long laboratory evolut ionary exper iments with E. coli BW25II3. T h e used strains were initially sensitive to antibiotics and di f fer only in their respective consti tutive genomic mutat ion rates (wild type BW25II3 versus AmutS) and/or activities of the SOS response (wild type versus L e x A 3 expressing strain or AmutSLexA3) . % independent l ineages of each bacterial strain were al lowed to evolve in microti ter plates to successively higher antibiotic concentrat ions by t ransferr ing daily 1% of each cul ture. We employed three antibiotics: c iprof loxacin, ampicil l in. tobramycin, representing three major c lasses of bactericidal ant ibiot ics (quinolones . P- lactams and aminoglycosides respectively) known to stimulate the product ion of ROS. As a negat ive control , w e a lso tested the bacter io-static t r imethoprim, a folic acid biosynthesis inhibitor. In order to test the contribution of ROS format ion on evolvability we added thiourea to the medium. Thiourea is a hydroxyl radical scavenger which mitigates the damage caused by ROS format ion upon antibiotic t rea tment . 206 Volume 5S(1):197-212, 2011 Acta Biologica Szegediensis http: / /www.sci .u-szeged.hu/ABS We found lhat thiourea substantially promotes extinction of evolving bacterial populations for all three cases of bactericid antibiot-ics treatments both in the case of mutators and non-mutators. Our results indicate that scavenging of reactive oxygen species by thiourea substantially reduces mutational input, and hence the corresponding populations had lower opportunity to evolve resistance against the used antibiotic. Thiourea treatment had no significant impact on extinction rates of the populations adapted to the bacteriostatic drug trimethoprim. We found that the impact of ROS formation on resistance evolution is at least partially independent of the SOS response. Our work suggests that enhancement of ROS formation may be useful for improving antibiotics lethality in the short term, but it also ac-celerates drug resistance evolution. Supervisor: Csaba Pál E-mail: Investigation of a novel stress-induced operon in Synechocystis PCC 6803 Csaba István Nagy Laboratory o f Molecu lar Stress and Photobio logy, Inst i tu te o f Plant Biology, Biological Research Center, Hungar ian Academy o f Sciences, Szeged, Hungary Cyanobacteria are photosynthetic prokaryotes that are widely used model organisms of photosynthetic research according to their close relationship to chloroplasts. These prokaryotes possess a variety of adaptation mechanism by which they are able to inhabit different ex-treme biotops. Since they are autotrophic organisms they are the primary producers of these niches. Among others, arsenic compounds and hydrogen sulfide are widely distributed in nature since the origin of life. Cyanobacteria have evolved mechanisms to cope with the toxicity of these substances. In order to maintain their homeostasis under fluctuating environmental changes cyanobacteria evolved environmentally inducible detoxification systems. We investigated the expression pattern of different genes upon heavy metal treatment in Synechocystis sp. PCC 6803 and discovered a novel gene cluster that responds to As3 ' . This operon is located on the pSYSM plasmid and contains four genes, termed artRSCT. ArtR and artS are transcribed in the opposite direction of that of artC and artT, with a common promoter region between these two tandem gene pairs. The gene product of artR shows high homology with DNA-binding proteins of the ArsR family. We have constructed a deletion mutant in order to inactivate the artR gene. In this mutant we observed high constitutive expression of the artS/CfT genes using quantitative RT-PCR technique. This fact supported our hypothesis that it functions as a common repressor of the genes. We have cloned and expressed this protein in E. coli in order to obtain further proof of its function. Using this purified protein we performed an electrophoretic mobil-ity shift assay, which confirmed the specifity of ArtR towards the promoter sequence. Taken all these results into consideration we have proven that the protein product of artR is a regulator of the artRSCT operon, which binds to the common promoter and dissociates in the presence of As**. ArtT shows high homology with membrane-bound heavy metal transporter proteins and because it is induced by As'*, we assumed that it may be part of an arsenic resistance system. In order to test this hypothesis we have constructed an artT insertional mutant. The results were contradictory to our hypothesis because the mutant was more resistant to elevated amounts of As5* then the wild type. Thus the func-tion of artT may be different from that of the arsenic resistance genes. The protein product of artS gene is homologous to sulfide-quinone oxido-reductases (SQR) and shows a high expression not only to As5* but to Na,S as well. Moreover Synechocystis PCC 6803 was more resistant to high amounts of Na,S then other cyanobacterial species lhat lacks the SQR enzyme. H,S and As5* can serve as alternative electron donors in many ancient anaerobic photosynthetic systems. The procaryotes capable of this type of electron transport use a single photosystem (PSI) and harbor a SQR enzymes, which oxidize sulfide to sulfur. Other strains can use arscnite oxidase enzyme to grow phototrophically in As5* containing anaerobic environment. The facts that the artRSCT genes are induced by As5* and Na,S and the strain is more tolerant to Na,S, than those which lack such genes suggest that these genes, especially artS may be involved in sulfide tolerance or sulfide metabolism of the cyanobacterium. Supervisors: Péter Kós. Imre Vass E-mail: ncsaba@brc.hu 207 

The role of oxidative stress in antibiotic resistance evolution

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The role of oxidative stress in antibiotic resistance evolution

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