Научные стремления 2011-1

6.5 at 30ºC and Km of 0.21 mM and 10.5 mM for o-nitrophenyl-β-D- galactopyranoside and lactose, respectively. It was supposed that beta-galactosidase consists of a basic tetrameric form (4 N) together with minor quantities of other higher aggregate forms up to 16 N. The enzyme of Arthrobacter sp. is less stable to heat and urea exposure than Escherichia coli enzyme [9]. According to Erickson (1970) opinion, it is explained by relationship between the existence of multiple forms and stability of the basic tetramer, with the enzymes of greatest stability showing the greatest aggregate forms [10].

Loveland et al. (1994) screened out psychrotrophic microorganism with betagalactosidase activity from Pennsylvanian (USA) fields that had been spread with whey during winter. This isolate grown within temperature range from 0 to 30ºC was identified as Arthrobacter sp. B7 [11]. All its beta-galactosidase activity was associated with the cells and revealed temperature optimum about 20°C below that of

Escherichia coli.

At least two proteins that hydrolyzed specific substrate 5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside were found in isolate B7 cell-free extracts by nondenaturing electrophoresis. To determine whether the activities were due to different isoenzymes of Arthrobacter sp. B7 or were simply aggregates of the same protein, the effects of changing the polyacrylamide concentrations on the migration distance of the protein bands or the effects of the pH on its intensity were examined in situ. The intensities of the protein bands were established to vary independently of each other, suggesting that the beta-galactosidase activities originated from different proteins. Furthermore, the intensity of the upper enzyme band was influenced by growth conditions whereas the lower band was present even when lactose was substituted for glucose or cellobiose in cultural media. It was supposed that these proteins would not be true beta-galactosidases; they might be glucosidases or other enzymes with sufficiently broad specificities capable to hydrolyze the substrate slowly [11].

Further investigation showed that three different genes encode Arthrobacter sp. B7 beta-galactosidase. The major isozyme 15 has temperature optimum between 4550ºC, Km value for o-nitrophenyl-β-D-galactopyranoside and lactose is 0.4 and 2.5 mM, respectively. Another isoenzyme 12 reveals maximum activity at about

40ºC. It was further established that isoenzyme 12 has subunit about 71 kDa which is considerably smaller than that found for isoenzyme 15 (111 kDa) and other typical lacZ enzymes (116 kDa for Escherichia coli). The isozyme 14 – a product of third gene expression in cell-free extracts was not detected yet [12].

To determine whether the expression of isoenzyme 12 and isoenzyme 15 having slightly distinct temperature of action might be regulated differently, the original psychrotrophic strain Arthrobacter sp. B7 was grown at a low temperature

(4ºC) and a temperature near the maximum for growth (25ºC) [13]. It was found that bacterial cells demonstrate total beta-galactosidase activity when grown on medium with lactose equal to 0.92 and 1.84 µmol/mg of protein at 4 and 25ºC, respectively, with cellobiose – 0.05 µmol/mg of protein both at 4 and 25ºC, and with glucose – 0.09 and 0.03 µmol/mg of protein at 4 and 25ºC, respectively. Thus, the total enzyme

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activity was increased 20to 60-fold in cells grown with lactose as opposed to glucose and cellobiose.

Western blot analysis showed that namely isozyme 15 increased dramatically in lactose-grown Arthrobacter sp. B7, whereas isozyme 12 levels remained relatively constant in bacterial cells grown on media with all three saccharides. Thus, isozyme 15 is regulated as a typical lactose-utilizing enzyme; its intracellular levels increase in medium with lactose and decrease in media with either glucose or cellobiose. These results suggest that isozyme 15 is responsible for lactose utilization in isolate B7 under the studied growth conditions. In contrast, it is not clear whether isozyme 12 play a direct role in lactose metabolism in bacterium, however, Escherichia coli transformants expressing this protein form colonies on media containing lactose as a carbon source. Perhaps, isozyme 12 functions during rapid temperature changes promoting Arthrobacter sp. B7 adaptation [13].

Following examination of chromosomal DNA from Arthrobacter sp. B7 showed that it harbors gene designated as 14.2. This gene has homology with the human acid beta-galactosidase and encodes a subunit of 52 kDa. The enzyme appears to be active as a dimer and hydrolyzes substrates with either a β-1,4- or β-1,3- glycoside linkage. The enzyme purified by affinity chromatography with p- aminophenyl-β-D-thiogalactopyranoside has pH optimum at 6.5 and remains stable for over 2 h at or below 30ºC, but is inactivated at 50ºC [14].

From Antarctic Dry Valley region strain Arthrobacter sp. SB was isolated. Its cell-free extract reveals two peaks of beta-galactosidase activity: one peak near 15°C and another around 35°C, suggesting the presence of more than one enzyme [15]. Gene bgaS encoding a family 2 beta-galactosidase was isolated from Arthrobacter sp. SB and cloned in Escherichia coli. Expressed enzyme displays maximum activity at

18ºC and retains 50% of that at 0°C. The results of analyzing its oligomeric state by analytical ultracentrifugation showed that enzyme is a tetramer (approximately 463 kDa) at 4°C, where it is active, and is dissociating into monomer (112 kDa) at 25°C.

Nakagawa et al. (2003) isolated from soil of Hokkaido (Japan) five strains F1, F2, F3, F4 and F5 of Arthrobacter psychrolactophilus growing under 5°C on media with lactose as a sole carbon source and showing highly specific beta-galactosidase activity even at 0°C [16]. The enzyme exhibits the highest catalytic activity at 10°C, and loses less than 20% of maximum activity at 0°C. Using zymogram analysis it was shown that intracellular beta-galactosidase of A. psychrolactophilus strains F1, F2, F3 and F5 grown on lactose-containing media gives only one activity band, with the same relative mobility (Rm) at 5°C. However, strains F1, F3 and F5 give a second activity band at 30°C. These results show that above-mentioned strains have at least two beta-galactosidase isozymes, and it seems that the one with the small Rm value is a cold-active beta-galactosidase, and the other with the large Rm value is a normal beta-galactosidase. Strain F4 possesses another type of beta-galactosidase, having a different Rm value from enzyme of other strains.

As a result of further research it was established that Arthrobacter psychrolactophilus strain F2 has only one type of intracellular cold-active beta-

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galactosidase encoded by bglA gene. This enzyme is probably a tetramer (548 kDa) with temperature and pH optima at 10°C and 8.0, respectively [17].

Nakagawa et al. (2007) reported superproduction of beta-galactosidase from

A. psychrolactophilus F2 using Escherichia coli cold gene expression system. The recombinant enzyme (rBglAp) hydrolyzes lactose as well as o-nitrophenyl-β-D- galactopyranoside (20.4 U/mg at 10°C), and exhibits a high specific activity at 0°C, similar to the native beta-galactosidase from A. psychrolactophilus F2. Moreover, a product corresponding to trisaccharide was detected in the reaction mixture, indicating the rBglAp also exhibits transglycosylation activity [18].

Strain C2-2 of Antarctic bacterium Arthrobacter sp. produces two, possibly three cold-active isoenzymes of beta-galactosidase. According to native electrophoresis data, bacterium synthesizes two catalytically active proteins – isoenzyme C2-2-2 with large Rm value and isoenzyme C2-2-1 with small Rm value when grown on complete or on minimal medium containing 3% lactose, respectively. Isoenzyme C2-2-3 could not be detected in situ using the activity assay with 5- bromo-4-chloro-3-indolyl-3-D-galactopyranoside as the substrate, and it did not cleave lactose [19]. The isoenzyme C2-2-1 induced by lactose has a lower temperature optimum (25°C) and a higher proportion of beta-galactosidase activity at

10°C (42%) than its counterpart C2-2-2. It also showed higher specific activity on lactose; ratio of C2-2-1 affinity to o-nitrophenyl-β-D-galactopyranoside and lactose is 3:1, versus 24:1 in C2-2-2 [20].

The C2-2-1 isoenzyme resulting from Arthrobacter sp. C2-2 beta-galactosidase gene expression in Escherichia coli is a homotetramer of approximately 550 kDa size with a subunit molecular weight of 111 kDa. The enzyme optima for lactose hydrolysis were found to be 40oC and pH 7.5. Yet, the isoenzyme C2-2-1 has transglycosylation activity and forms triand tetrasaccharides [19].

The X-ray structure of isoenzyme C-2-2-1 from strain C2-2 of Arthrobacter sp. was solved at 1.9 resolution. It was shown that the enzyme forms 660 kDa hexamers, molecular mass of subunit is 110 kDa [21].

A psychrotrophic bacterium Arthrobacter sp. 20B producing intracellular coldadapted beta-galactosidase was isolated from the soil sampled in the neighborhood of Henryk Arctowski Polish Antarctic Station at King George Island [22]. The number of beta-galactosidase isozymes was determined by method of zymograms in the cellfree extracts of Arthrobacter sp. 20B. When this bacterium was grown on medium with lactose, only one protein band was visible on the zymograms, providing evidence that the strain produces only one molecular form of beta-galactosidase. The enzyme displays much lower activity during hydrolysis of its natural substrate lactose as compared to the cleavage of such synthetic substrates as o-nitrophenyl-β-D- galactopyranoside or p-nitrophenyl-β-D-galactopyranoside, preferring the latter substrate. The beta-galactosidase from Arthrobacter sp. 20B reveals maximum activity at pH 6.0-8.0 and 25°C; retains stability up to 35°C for 60 min, but apparently loses activity at 10°C. Beta-galactosidase is significantly activated by 2- mercaptoethanol and dithiotreitol and inhibited by pCMB. This effect provides

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evidence that free thiol groups of cysteine residues are necessary for activity of betagalactosidase, but the role of these groups in catalysis remains to be deciphered.

Arthrobacter sp. 32c was isolated from the Antarctic soil. The gene encoding beta-galactosidase of this bacterium has been isolated, sequenced, cloned, and expressed in Escherichia coli and Pichia pastoris. E. coli expression systems produce enzyme intracellularly and P. pastoris – extracellularly. The enzyme was found to be active as a homotrimeric protein of 195 kDa, each monomer is 75.9 kDa. There is no evidence of multiple molecular forms or isozymes of Arthrobacter sp. 32c betagalactosidase. The recombinant enzyme shows the highest activity at pH 6.5 and

50°C and displays four times higher level of activity with p-nitrophenyl-β-D- galactopyranoside than with o-nitrophenyl-β-D-galactopyranoside as the substrate [23].

Lately strains Arthrobacter sp. BIM B-2239, BIM B-2240, BIM B-2241 and BIM B-2242 producing truly extracellular beta-galactosidase were screened out among cultures from Belarusian Collection of Nonpathogenic Microorganisms. The ratio of cell-bound enzyme does not exceed 0.1-0.2% of total enzyme protein synthesized by the bacteria [24]. These strains grown on media either with lactose or readily metabolized carbon sources produce beta-galactosidase in different quantities. Taking into consideration this fact existence of multiple molecular forms of the enzyme protein in studied Arthrobacter sp. strains is expected.

Partly purified beta-galactosidase from cell-free cultural liquid of the most active strain Arthrobacter sp. BIM B-2242 displays maximal catalytic activity at pH

7.0 and temperature 42°C, and retains stability at 30°C and 40°C in pH range of 6.0- 8.0 and 6.0-7.0, respectively, at least for 15 min. Michaelis constant of the enzyme with respect to o-nitrophenyl-β-D-galactopyranoside under optimal conditions is 27 mM [25]. Studies of Arthrobacter sp. BIM B-2242 beta-galactosidase having unique extracellular localization are planned using physiological, biochemical, genetic and molecular biology methods.

Summing up above-mentioned literature data, members of Arthrobacter genus produce intracellular beta-galactosidases represented in most cases by forms differing in molecular weight, affinity to specific substrate and physical-chemical properties. Only in rare publications there is strict evidence of its genetical, not posttranslational origin. Probably, the existence of isozymes with different properties in Arthrobacter bacteria is the mechanism that allows them to adapt to widely varying environmental habitat. For instance, various temperature optima of beta-galactosidase molecular forms provide for microorganism a competitive advantage under rapidly changing temperature conditions over the species lacking such enzymes.

In conclusion, data on beta-galactosidase activity of Arthrobacter bacteria, molecular composition of produced enzyme complexes, its genesis, biological significance, substrate specificity, and biochemical and catalytical properties of the enzyme protein are fragmentary and contradictory. It determines key significance of this research trend supported by vital role of microbial beta-galactosidase in manufacturing functional food and fodder from whole milk and its processing wasters.

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Литературные источники

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2.Panesar, P. S. Potential applications of immobilized β-galactosidase in food processing industries / P.S. Panesar, S. Kumari, R. Panesar // Enzyme Res. – 2010. – Vol. 2010. – P. 1-16.

3.Husain, Q. β-Galactosidases and their potential applications: a review / Q. Husain // Crit. Rev. Biotechnol. – 2010. – Vol. 30, № 1. – P. 41–62.

4.Lactulose biosynthesis by β-galactosidases from a newly isolated Arthrobacter sp. / L. Tang [et al] // J. Ind. Microbiol. Biotechnol. – 2011. – Vol. 38. – P. 471–476.

5.Secreted beta-galactosidase from a Flavobacterium sp. isolated from a lowtemperature environment / H.P. Sorensen [et al.] // Appl. Microbiol. Biotechnol. – 2006. – Vol. 70.

– P. 548–557.

6.Heterologous expression of a gene encoding a thermostable β-galactosidase from

Alicyclobacillus acidocaldarius / T. Yuan [et al.] // Biotechnol. Lett. – 2008. – Vol. 30. – P. 343– 348.

7.Asraf, S. S. Current trends of β-galactosidase research and application / S.S. Asraf, P. Gunasekaran // Cur. Res. Tech. Educ. Topics in Appl. Microb. Microb. Biotech. – 2006. – Vol. 70. – P. 548–557.

8.Nomenclature of Multiple Forms of Enzymes // IUPAC-IUB Commission on Biochemical Nomenclature (CBN) [electronic resource]. - Mode of access: http://www.chem.qmul.ac.uk/iubmb/misc/isoen.html. - Date of access: 10.10.2011.

9.Donnelly, W. J. Some properties of a multiple-form β-galactosidase from an Arthrobacter sp. / W.J. Donnelly, I.N. Fhaolain // Int. J. Biochem. –1977. – Vol. 8. – P. 101–106.

10.Erickson, R.P. Comparative study of isoenzyme formation of bacterial β- galactosidase / R.P. Erickson, E.S. Steers // J. Bacteriol. – 1970. – Vol. 102, № 1. – P. 79–84.

11.Characterization of psychrotrophic microorganisms producing β-galactosidase activities / J. Loveland [et al.] // Appl. Environ. Microbiol. – 1994. – Vol. 60, № 1. – Р. 12–18.

12.Characterization of a psychrotrophic Arthrobacter gene and its cold-active β-

galactosidase / D.E Trimbur [et al.] // Appl. Environ. Microbiol. – 1994. – Vol. 60, № 12. – Р. 4544–4552.

13.Analysis pf a novel gene and β-galactosidase isozyme from a psychrotrophic Arthrobacter isolate / K.R. Gutshall [et al.] // J. Bacteriol. – 1995. – Vol. 177, № 8. – P. 1981–1988.

14.Gutshall, K. A novel Arthrobacter β-galactosidase with homology to eukaryotic β- galactosidases / K. Gutshall, K. Wang, J.E. Brenchley // J. Bacteriol. – 1997. – Vol. 179, No. 9. – P. 3064-3067.

15.Biochemical characterization of a β-galactosidase with a low temperature optimum obtained from Antarctic Arthrobacter isolate / J.A. Coker [et al.] // J. Bacteriol. – 2003. – Vol. 185, № 18. – P. 5473–5482.

16.Isolation and characterization of psychrophiles producing cold-active β-galactosidase / T. Nakagawa [et al.] // Lett. Appl. Microbiol. – 2003. – Vol. 37. – P. 154–157.

17.Purification and molecular characterization of cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2 / T. Nakagawa [et al.] // Appl. Microbiol. Biotechnol. – 2006. – Vol. 72. – P. 720–725.

18.Overexpression and functional analysis of cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2 / T. Nakagawa [et al.] // Protein Expr. Purific. – 2007. – Vol. 54. – P. 295–299.

19.The cloning, purification and characterization of a cold-active β-galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp. C2-2 / P. Karasová-Lipovová [et al.] // Enzyme Microbial. Technol. – 2003. – Vol. 33. – P. 836–844.

20.Beta-galactosidase activity in psychrotrophic microorganisms and their potential use in food industry / P. Karasova [et al.] // Czech. J. Food Sci. – 2002. – Vol. 20, № 2. – P. 43–47.

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21.Cold-active β-galactosidase form Arthrobacter sp. C2-2 forms compact 660 kDa

hexamers: crystal structure at 1.9 Ả resolution / T. Skalova [et al.] // J. Mol. Biol. – 2005. – Vol. 353. – P. 282–294.

22.A new β-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization / A.M. Bialkowska [et al.] // Arch. Microbiol. – 2009. – Vol. 191. – P. 825–835.

23.Hildebrandt, P. A new cold-adapted beta-D-galactosidase from the Antarctic Arthrobacter sp. 32c - gene cloning, overexpression, purification and properties / P. Hildebrandt, M. Wanarska, J. Kur // BMC Microbiology. – 2009. – Vol. 9. – P. 1–11.

24.β-Galactosidase activity in actinobacteria of Arthrobacter genus / A. Kostenevich [et al.] // Modern problems of microbiology and biotechnology: the young scientists' and students' international scientific conference: book of abstracts, Odesa, 28-31, May 2007 y. / Odesa National University; editor-in-chief: К.О. Ivanytsya. – Odesa, 2007. – P. 74.

25.Characterization of extracellular β-galactosidase produced by Arthrobacter sp. B- 2242 / L.I. Sapunova [et al.] // Microbial diversity: current situation, conservation strategy and biotechnological potential: Proc. III Int. conf., Perm, 28 September – 5 October 2008 y. / Institute of Ecology and Genetics of Microorganisms, Ural Branch, Russian Academy of Sciences; exec. edit.: I.B. Ivshina [et al.]. – Perm, 2008. – P. 195-196.

Kastsianevich A.A., Sapunova L.I.

BETA-GALACTOSIDASES OF ARTHROBACTER BACTERIA: MULTIPLE FORMS OR ISOENZYMES?

Institute of Microbiology, Belarus National Academy of Sciences, Minsk

Summary

Literature data related to beta-galactosidase activity of Arthrobacter genus bacteria, enzyme localization, its structure, substrate specificity, and physical-chemical properties were summarized.

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УДК 634.11:632.35

Л.Л. Лагоненко, А.Л. Лагоненко

КОНСТРУИРОВАНИЕ И ХАРАКТЕРИСТРИКА ДЕЛЕЦИОННОГО МУТАНТА ERWINIA AMYLOVORA ПО СИСТЕМЕ СЕКРЕЦИИ III ТИПА

Белорусский государственный университет, Минск

Введение. Erwinia amylovora, представитель семейства Enterobacteriaceae, является возбудителем бактериального ожога, тяжелого заболевания, поражающего яблони, груши и другие растения семейства Rosaceae и причиняющего огромный ущерб садам по всему миру. К факторам патогенности Erwinia amylovora относятсятся субстраты системы секреции III типа (T3SS), экзополисахариды (ЭПС) амиловоран и леван [1]. ЭПС в совокупности с поверхностными белками клеток вовлечены в формирование биопленки, образуемой E. amylovora. Формирование биопленки помогает одноклеточным организмам формировать мультиклеточное сообщество. Преимущества такого сообщества заключаются в том, что бактерии становятся менее уязвимыми к изменениям условий окружающей среды, действию антибиотиков и защитным системам растений [2]. Столь большое разнообразие различных факторов вирулентности бактерий E. amylovora позволяет предполагать наличие какой-то общей системы регуляции их синтеза. Понимание молекулярных механизмов патогенности и вирулентности E. amylovora поможет в дальнейших исследованиях по разработке эффективных методов борьбы с бактериальным ожогом. Данная работа посвящена получению и последующей характеристике делеционного мутанта E. amylovora по системе секреции III типа.

Материалы и методы. Конструирование мутанта. Для получения делеционного мутанта E. amylovora по T3SS была использована техника «PCRbased one-step inactivation of chromosomal genes» [3]. В работе были использован штамм E. amylovora 1/79, плазмиды pKD46 (экспрессирует α-, β- и γ- рекомбиназу) и pKD13 (содержит ген KmR), праймеры T3_F1, T3_R1, Km1, Km2, CT3_F1, CT3_R1 [4]. Для подтверждения делеции T3SS, проводили ПЦР с парой праймеров, комплементарных концевым областям T3SS, и еще одной парой, комплементарной внутренной области KmR-гена.

Тесты на патогенность. Проверка патогенности делеционного мутанта проводилась на цветках и незрелых плодах груши по стандартной методике [4]. Способность индуцировать реакцию гиперчувствительности клетками E. amylovora оценивали путем инфильтрации в листья табака и бобов.

Тесты на подвижность. Для проведения тестов на подвижность бактерий использовалась минимальная среда M9 и созданная на ее основе hrpиндуцирующая среда с лимитом по источнику азота, содержащая 0,3% агара.

Тест на формирование биопленки на стекле. Для оценки способности мутанта формировать биопленку in vitro использовался метод, описанный у Koczan et al. 2009 [2], с изменениями.

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Результаты и обсуждение. На первом этапе работы нами был сконструирован делеционный мутант E. amylovora по системе секреции III типа. Клетки бактерий E. amylovora 1/79 подвергали мутагенезу по методу Даценко и Воннера. В результате проделанной работы был отобран штамм (далее LTS-3), устойчивый к канамицину. Для подтверждения наличия делеции системы секреции третьего типа, ДНК из клеток E. amylovora LTS-3 амплифицировали с праймерами к областям, фланкирующим делецию (СT3_F1 и CT3_R1), а также с внутренними праймерами к гену устойчивости к канамицину (Km1 и Km2). В результате ПЦР были получены фрагменты ДНК ожидаемых размеров (рисунок 1). Полученный мутант оказался не способным индуцировать симптомы бактериального ожога при искусственном введении в

плоды и цветки груши Клетки E. amylovora LTS-3 не вызывали развитие реакции гиперчувствительности в листьях табака и бобов. Авирулентность и не способность вызывать реакцию гиперчувствительности являются четкими признаками наличия мутаций по системе секреции третьего типа в клетках бактерий E. amylovora.

Рисунок 1- Электрофореграмма продуктов амплификации ДНК из клеток E. amylovora LTS-3 (дорожки 1-3) и 1/79 (дорожки 4-6) с праймерами R1/Km1(дорожки 1 и

4), F1/Km2 (дорожки 2 и 5) и F1/R1 (дорожки 3 и 6). М – GeneRuler 1 kb DNA Ladder (Fermentas)

До недавнего времени считалось, что в состав HrpL-регулона бактерий E. amylovora входят лишь гены, кодирующие структурные компоненты системы секреции третьего типа и гены эффекторов – харпинов и Avr-белков. Однако после расшифровки и тщательного анализа генома этого фитопатогена промоторы альтернативного сигма-фактора HrpL были выявлены перед множеством других генов, не связанных с ССТТ. Учитывая тот факт, что полученный нами штамм несет делецию всего hrp-кластера, включая ген hrpL, было интересно изучить эффект такой мутации на другие факторы вирулентности E. amylovora, в особенности на подвижность бактерий, продукцию экзополисахаридов и способность образовывать биопленки. Известно, что индукция альтернативного сигма-фактора HrpL происходит при выращивании бактерий в минимальных питательных средах с лимитом по источнику азота. Поэтому тесты по оценке подвижности клеток E. amylovora LTS-3 осуществляли на минимальных питательных средах с нормальным и лимитированным содержанием азота. При выращивании E. amylovora на минимальной среде М9 значимых различий в подвижности клеток мутанта и

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исходного штамма не наблюдалось, тогда как в условиях дерепрессии системы секреции третьего типа подвижность клеток LTS-3 возрастала в 1,5 раза по сравнению с контролем. Полученные результаты косвенно указывают на возможность негативной регуляции подвижности клеток E. amylovora альтернативным сигма-фактором HrpL.

На следующем этапе работы была осуществлена оценка способности клеток штаммов E. amylovora LTS-3 и 1/79 формировать биопленки на поверхности стеклянных пластинок, погруженных в бактериальную суспензию (рисунок 2).

Рисунок 2 - Биопленки на поверхности стеклянных пластинок, сформированные клетками E. amylovora 1/79 (А) и LTS-3 (В)

Как видно из рисунка 2, клетки LTS-3 в значительно худшей степени адсорбировались на поверхности пластинок и не формировали биопленку. Такое фенотипическое проявление мутации указывает на взаимосвязь между наличием функциональной системы секреции третьего типа и способностью формировать биопленку клетками E. amylovora. Способность бактериальных клеток эффективно адсорбироваться на различных поверхностях во многом зависит от продукции экзополисахаридов. Поэтому на следующем этапе нами было оценено количество внеклеточных полисахаридов, синтезируемых мутантными и исходными клетками E. amylovora. Как ни странно, клетки E. amylovora LTS-3 продуцировали значительно большее количество ЭПС, по сравнению с 1/79. Полученные данные подтверждают гипотезу о значительно более разнообразном составе HrpL-регулона и позволяют предположить, что система регуляции секреторного аппарата третьего типа может напрямую или опосредованно взаимодействовать с другими сигнальными путями для координации экспрессии генов в процессе патогенеза.

Литературные источники

1.Vanneste, J.L. 2000. Fire Blight, the disease and its causative agent, Erwinia amylovora. CABI Publ. pp.370.

2.Koczan, J.M., McGrath, M.J., Zhao Y., and Sundin, G.W. 2009. Contribution of Erwinia amylovora exopolysaccharides amylovoran and levan to biofilm formation: implications in pathogenicity. Phytopathology 99:1237-1244

3.Datsenko, K.A., and Wanner, B.L. 2000. One step inactivation of chromosomal genes in Escherichia coli K-12 using PCR-products. Proc. Natl. Acad. Sci. USA 97:6640-6645.

4.Zhao, Y., Sundin, G.W., Wang D. 2009 Construction and analysis of pathogenicity island deletion mutants of Erwinia amylovora Can J. Microbiol. 55: 457-464.

5.Romling, U. Molecular biology of cellulose production in bacteria. Research in Microbiology, 2002.153(4):p. 205-212.

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L.L. Lagonenko, A.L. Lagonenko

CONSTRUCTION AND CHARACTERIZATION OF ERWINIA AMYLOVORA TYPE III SECRETION SYSTEM DELETION MUTANT

Belarusian State University, Minsk

Summary

Erwinia amylovora is the causal agent of fire blight, a devastating necrotic disease of rosaceous species. It possesses a Type III secretion system (T3SS) that functions in the delivery of effector proteins straight into the host plant cell. In this work we constructed its Type III secretion system deletion mutant using one-step inactivation of chromosomal genes using PCR-products technique and studied it characteristics including pathogenicity, motility, exopolysaccharides and biofilm formation.

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