The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype

Aim. To estimate WaaL ligase contribution in the lipopolysaccharide (LPS) phenotype profile formation of Y. enterocolitica O:3 (YeO3) bacteria. Methods. The waaL-knock-out mutants were created by an allelic exchange strategy. The LPS phenotypes of created mutants were visualized by silver-stained DO...

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Дата:	2014
Автори:	Shevchenko, J.I., Pozur, V.K., Skurnik, M.
Формат:	Стаття
Мова:	English
Опубліковано:	Інститут молекулярної біології і генетики НАН України 2014
Назва видання:	Вiopolymers and Cell
Теми:	Structure and Function of Biopolymers
Онлайн доступ:	http://dspace.nbuv.gov.ua/handle/123456789/154571
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Назва журналу:	Digital Library of Periodicals of National Academy of Sciences of Ukraine
Цитувати:	The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype / J.I. Shevchenko, V.K. Pozur, M. Skurnik // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 443-447. — Бібліогр.: 8 назв. — англ.

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spelling	irk-123456789-1545712019-06-16T01:31:30Z The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype Shevchenko, J.I. Pozur, V.K. Skurnik, M. Structure and Function of Biopolymers Aim. To estimate WaaL ligase contribution in the lipopolysaccharide (LPS) phenotype profile formation of Y. enterocolitica O:3 (YeO3) bacteria. Methods. The waaL-knock-out mutants were created by an allelic exchange strategy. The LPS phenotypes of created mutants were visualized by silver-stained DOC-PAGE and immunoblotting with specific outer core (core oligosaccharide, hexasaccharide, OC) and O-polysaccharide (OPS or O-Ag) monoclonal antibodies. Results. Deletion of waaLOS gene from YeO3 genome has a marked effect on OC ligation in either single or double mutants. The waaLPS deletion has an opposite effect on the OPS ligation – barely detected increasing of OPS bands. Conclusions. The LPS ligases of YeO3 exhibit relaxed donor substrate specificity. Under given conditions the effect of WaaLOS ligase is more significant for OC and OPS ligation onto lipid A than that of WaaLPS. Мета. Дослідити участь лігаз WaaL у формуванні фенотипу ліпополісахариду (LPS) серед бактерій Y. enterocolitica O:3 (YeO3). Методи. Нокаутні мутанти по генах лігаз waaL створено внаслідок обміну алелями. Фенотипи LPS отриманих мутантів візуалізували, забарвлюючи сріблом гель DOC-PAGE, а також використовували імуноблот зі специфічними моноклональними антитілами до кору (корового олігосахариду, гексасахариду, ОC) та О-полісахариду (OPS, O-Ag). Результати. Делеція гена лігази waaLOS з геному бактерій YeO3 чинить помітний вплив на лігування ОC як в одиночних, так і в подвійних мутантах. Проте маніпуляції з геном лігази waaLPS призводять до ледь помітної стимуляції утворення OPS. Висновки. Лігази LPS бактерій YeO3 демонструють низьку субстратну специфічність. Участь лігази WaaLOS у формуванні повноцінної структури LPS є суттєвішою, аніж WaaLPS, за даних умов. Цель. Исследовать участие лигаз WaaL в формировании фенотипа липополисахарида (LPS) среди бактерий Y. enterocolitica O:3 (YeO3). Методы. Нокаутные мутанты по генам лигаз waaL созданы вследствие обмена аллелями. Фенотипы LPS полученных мутантов визуализировали, окрашивая серебром гель DOC-PAGE, а также с использованием иммуноблота со специфическими моноклональными антителами к кору (коровому олигосахариду, гексасахариду, OC) и О-полисахариду (OPS, O-Ag). Результаты. Делеция гена лигазы waaLOS из генома бактерий YeO3 оказывает заметное влияние на лигирование OC как в одиночных, так и двойных мутантах. Однако манипуляции с геном лигаз waaLPS приводят к едва заметной стимуляции образования OPS. Выводы. Лигазы LPS бактерий YeO3 демонстрируют низкую субстратную специфичность. Участие лигазы WaaLOS в образовании полноценной структуры LPS является более существенным, чем WaaLPS, при данных условиях. 2014 Article The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype / J.I. Shevchenko, V.K. Pozur, M. Skurnik // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 443-447. — Бібліогр.: 8 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008BE http://dspace.nbuv.gov.ua/handle/123456789/154571 579.234 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Structure and Function of Biopolymers Structure and Function of Biopolymers
spellingShingle	Structure and Function of Biopolymers Structure and Function of Biopolymers Shevchenko, J.I. Pozur, V.K. Skurnik, M. The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype Вiopolymers and Cell
description	Aim. To estimate WaaL ligase contribution in the lipopolysaccharide (LPS) phenotype profile formation of Y. enterocolitica O:3 (YeO3) bacteria. Methods. The waaL-knock-out mutants were created by an allelic exchange strategy. The LPS phenotypes of created mutants were visualized by silver-stained DOC-PAGE and immunoblotting with specific outer core (core oligosaccharide, hexasaccharide, OC) and O-polysaccharide (OPS or O-Ag) monoclonal antibodies. Results. Deletion of waaLOS gene from YeO3 genome has a marked effect on OC ligation in either single or double mutants. The waaLPS deletion has an opposite effect on the OPS ligation – barely detected increasing of OPS bands. Conclusions. The LPS ligases of YeO3 exhibit relaxed donor substrate specificity. Under given conditions the effect of WaaLOS ligase is more significant for OC and OPS ligation onto lipid A than that of WaaLPS.
format	Article
author	Shevchenko, J.I. Pozur, V.K. Skurnik, M.
author_facet	Shevchenko, J.I. Pozur, V.K. Skurnik, M.
author_sort	Shevchenko, J.I.
title	The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype
title_short	The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype
title_full	The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype
title_fullStr	The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype
title_full_unstemmed	The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype
title_sort	effect of waal genes deletion from yersinia enterocolitica o:3 genome on bacteria lps’ phenotype
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2014
topic_facet	Structure and Function of Biopolymers
url	http://dspace.nbuv.gov.ua/handle/123456789/154571
citation_txt	The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype / J.I. Shevchenko, V.K. Pozur, M. Skurnik // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 443-447. — Бібліогр.: 8 назв. — англ.
series	Вiopolymers and Cell
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fulltext	UDC 579.234 The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype J. I. Shevchenko1, 2, V. K. Pozur1, M. Skurnik2 1Educational and Scientific Center «Institute of Biology» Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., Kyiv, Ukraine, 01601 2Haartman Institute, University of Helsinki Haartmaninkatu 3 (P. O. Box 21), Finland, FIN-00014 julia.i.shevchenko@gmail.com Aim. To estimate WaaL ligase contribution in the lipopolysaccharide (LPS) phenotype profile formation of Y. en- terocolitica O:3 (YeO3) bacteria. Methods. The waaL-knock-out mutants were created by an allelic exchange strategy. The LPS phenotypes of created mutants were visualized by silver-stained DOC-PAGE and immunoblot- ting with specific outer core (core oligosaccharide, hexasaccharide, OC) and O-polysaccharide (OPS or O-Ag) monoclonal antibodies. Results. Deletion of waaL OS gene from YeO3 genome has a marked effect on OC ligation in either single or double mutants. The waaL PS deletion has an opposite effect on the OPS ligation – barely detected increasing of OPS bands. Conclusions. The LPS ligases of YeO3 exhibit relaxed donor substrate spe- cificity. Under given conditions the effect of WaaL OS ligase is more significant for OC and OPS ligation onto lipid A than that of WaaL PS . Keywords: WaaL ligase, LPS, Yersinia enterocolitica, DOC-PAGE. Introduction. In Europe, yersiniosis is the third most common bacterial zoonosis after campylobacteriosis and salmonellosis [1]. Y. enterocolitica is a well known human and animal pathogen. Among humans, the path- way of Y. enterocolitica is associated with intestinal di- sease, such as enterocolitis, with inflammatory diarrhea, ileitis, mesenteric appendicitis and gastroenteritis. A di- arrheal disease is sometimes followed by post-infecti- ous reactive arthritis. It is well known that all Gram-negative bacteria con- tain an outer leaflet with a large amount of lipopolysac- charide (LPS). LPS is a glycolipid consisting of three domains: the lipid A moiety, the core and the distal O- polysaccharide (OPS or O-Ag). The homopolymeric O-Ag is composed of�1,2-linked 6-deoxy-L-altrose re- sidues. Together with the hexasaccharide core, the O- Ag is linked to the inner core (IC) of LPS to form a bran- ched structure [2]. The antigenic variations of OPS in the Y. enterocolitica isolates are distinguished serologi- cally. Nowadays, more than 50 serotypes are known, of which O:3, O:5, 27, O:8 and O:9 are pathogenic [3]. The LPS biosynthesis is a complex process that in- cludes the stepwise transformation of the primary sub- strate under enzymatic treatment. The WaaL proteins are involved in the ligation of OC and O-Ag onto the li- pid A core. According to the in silico investigations, the Y. enterocolitica O:3 (YeO3) genome contains at least three genes responsible for the WaaL proteins ex- pression. Yersinia pestis and Yersinia pseudotubercu- losis, however, carry only the waaLPS gene, whereas ei- ther waaLOS or waaLXS or both are additionally present in other Yersinia species. It was shown that deletion of waaLOS and waaLPS ge- nes correlate with the OPS and the OC expression. For this purpose LPS ligases were named as WaaLPS and WaaLOS respectively. The third ligase named as WaaLXS was not involved in the LPS or ESA biosynthesis [4]. The current study is aimed at the estimation of a ro- le of the ligases in the Y. enterocolitica’ LPS phenotype profile formation. The waaL-knock-out mutants of YeO3 443 ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 6. P. 443–447 doi: http://dx.doi.org/10.7124/bc.0008BE � Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014 444 were created by the allelic exchange strategy. Pheno- types of created mutants were visualized by silver-stai- ned DOC-PAGE and immunoblotting with specific OC and O-Ag monoclonal antibodies. Materials and methods. Bacterial strains and cul- ture conditions. Bacterial strains are listed in Table. Yersinia strains were grown at 22–25 oC (RT) and Es- cherichia coli strains at 37 oC in Luria Broth (LB) me- dia. LB supplemented with 1.5 % Bacto Agar was used for all solid cultures. As a selective medium CIN agar supplemented with appropriate antibiotics was used. When appropriate, the antibiotics were added to the me- dia at the following concentrations: kanamycin (Km), 100 µg/ml in agar plates and 20 µg/ml in broth; chlo- ramphenicol (Clm), 20 µg/ml. General DNA techniques. Isolation of plasmids and genomic DNA was done with kits. All enzymes were used according to the supplier’s specifications. Small- scale plasmid DNA preparations were carried out using plasmid mini prep kits. Plasmid DNA was moved by electroporation into Y. enterocolitica or heat shock trans- formation. Recombinant plasmids were mobilized from E. coli strains to Y. enterocolitica by conjugation. Mutants construction. The waaLOS and waaLPS ge- nes were amplified by PCR with primer pairs O3lig YE1727F5 & O3ligYE1727R5 and O3ligYE532F2 & O3ligYE532R2 with DyNAzyme DNA-polymerase («Thermo Scientific», USA) from isolated genomical DNA of YeO3. Amplified DNA was purified with Kit method and digested with NsiI (Mph 1103I) for the waaLOS gene and PstI for waaLPS. The digested and pu- rified fragments were cloned into the PstI digested suici- de vector pSW23T and the constructed plasmids were named as pSW23T-waaLOS and pSW23T-waaLPS respec- tively. The constructions were mobilized from E. coli �7249 into YeO3 strains by conjugation as described earlier [5]. For elimination of suicide vector and the wild- type genes, the optimized cycloserine enrichment me- thod was used [5]. For large-scale screening of knock-out mutants among Clm sensitive bacteria (ClmS) colonies we used Colony hybridization kit method («Roche», France). Isolated genomical DNA from negative colonies were diluted and used as a template for PCR with different primer pairs. DNA of wild-type strain YeO3 was used as a control. Complementation. The waaLOS and waaLPS genes were amplified with Phusion DNA polymerase from YeO3-c with O3ligYe1727f & O3ligYe1727r, O3lig Ye532f & O3ligYe532r primer pairs. PCR fragments were phosphorylated with polynucleotide kinase in the presence of 10 mM ATP, digested with EcoRI and liga- ted with EcoRI and ScaI digested, SAP-treated pTM100. The constructed plasmids were named pEPlig1727 & pEPlig532 and electroporated into S17-1� pir with fur- ther mobilization into YeO3 ligase mutants by conjuga- tion. Obtained colonies were screened on appropriate antibiotic plates with CIN agar [4]. SHEVCHENKO J. I. ET AL. Strain Genotype Reference Yersinia enterocolitica 6471/76 YeO3 wild type strain, patient isolate [6] 6471/76-c YeO3-c virulence plasmid cured derivative of 6471/76 [6] YeO3_�os waaLOS::pSW23Tlig1727su This work YeO3_�ps waaLPS::pSW29-lig532del, Km R This work YeO3_�os_�ps waaLOS::pSW23T-lig1727su waaLPS::pSW29-lig532del, Km R This work Escherichia coli �7249 B2163 �nic35, E. coli strain for suiside vector delivery, requirement for diaminopimelic acid 0.3 mM, Km R [7] S17-1�pir A-pir lysogen of S17-1, E. coli strain for suiside vector delivery [8] DH10B F-mcrA �(mrr-hsdRMS-mcrBC), �80lacZ�M15 �lacX74 recA1 endA1 araD139 �(ara, leu)7697 galU galK �-rpsL nupG tonA Life Tech- nologies Bacterial strains DOC-PAGE analysis. The bacteria were grown 16– 20 h at RT in 5 ml of LB medium with appropriate an- tibiotics. The exact optical density of the cultures was measured at 600 nm (OD600), 3 ml of the cultures were centrifuged and the pellets were resuspended in deoxy- cholate lysis buffer (2 % DOC, 4 % 2-mercaptoethanol, 10 % glycerol and 0.002 % bromophenol blue in 1 M Tris-HCl buffer, pH 6.8) in a volume adjusted accor- ding to density of the culture (100 l/OD600 ~ 1). Lypo- polysaccaride phenotypes were analyzed by silver stai- ned DOC-PAGE with previous proteinase K treated whole cell lysates [4]. The Western blotting O-polysac- charide, outer core and inner core expression were de- tected by O:3 specific mAbs 2B5 and TomA6. Results and discussion. Mutants construction. The waaLOS and waaLPS mutants of YeO3 were constructed from fully virulent serotype O:3 patient isolate expres- sing complete LPS. With help of the allelic exchange strategy we managed to inactivate waaLOS and waaLPS encoding regions in the YeO3 genome. The construc- ted single and double mutants were complemented with the pEPlig1727 & pEPlig532 plasmids, which were sup- plemented with the functional waaL gene. Traditional cycloserine enrichment method was ad- ditionally optimized [5]. The possibility was conside- red that constructed merodiploids (MD) are not fully re- sistant to Clm, as it should be (weak operon, etc.). To examine this possibility we tested several lines of condi- tions (concentration of Clm, incubation time, density of bacteria, etc.). According to these experimental data, the next improvements were done: reducing the Clm con- centration in media to 2.5 l v/v (instead of 10 l v/v) and prolongation of incubation to 4–5 h (instead of 2–3 h) before D-cycloserition solution was added. A current modification of the method allowed us to pick up only bacteria after second crossing over (Fig. 1). The colony hybridization method was used for spe- cific detection of deletion in the waaL gene among ClmS. Further justifications of deletion were performed by PCR (Fig. 2). Phenotype analysis. We used two approaches to analyze the difference in the expression of OC and O- Ag: 1) Silver staining of DOC-PAGE; 2) Immuno- blotting with OC and O-Ag specific antibodies. It is noticeable from silver staining of DOC-PAGE that deletion in the waaLOS gene leads to a dramatic de- crease in the OC expression and appearance of strong IC band (Fig. 3). However, the level of O-Ag expression reduced, as well, compared to the mutant with deletion in the waaLPS gene. The deletion in the waaLPS gene 445 THE EFFECT OF waaL GENES DELETION FROM Yersinia enterocolitica O:3 GENOME 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3.5 4.5Time, h O D 6 0 0 1 2 3 4 5 6 Fig. 1. Optimization of cycloserine en- richment method: 1 – YeO3 wild ty- pe (WT), 2.5 l v/v; 2 – WT, 5 l v/v; 3 – WT, 10 l v/v; 4 – merodiploids (MD), 2.5 l v/v; 5 – MD, 5 l v/v; 6 – MD, 10 l v/v 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Fig. 2. Amplification of waaL OS gene by PCR with pair of primers O3lig Ye1727f & O3ligYe1727r: 1–11 – checking colonies; 12 – gene ruler; 13 – waaL OS -knock-out mutant; 14 – YeO3 wild type control seems to be insignificant for the OC expression and even to stimulate the OPS formation. In case of the double YeO3_�os_�ps mutants, it is noticeable a strong ex- pression of IC bands, the absence of OC in both vari- ants and the absence of O-Ag expression in one variant. Western blotting analysis of LPS samples of the YeO3 ligase mutants and their complementation of sin- gle and double mutants were performed with the OC- specific mAb 2B5 and O-Ag-specific mAb TomA6 (Fig. 4). The deletion in the waaLOS gene resulted in re- duction of the OC expression, as it was shown with sil- ver staining of DOC-PAGE. Also, the waaLOS-knock- out mutant complemented with the functional waaLOS gene showed full recovery of OC and the decreasing of O-Ag expression at the same time. Similar results were obtained with the double mu- tant and complementation variants. The LPS profile of the double YeO3_�os_�ps mutant in immunoblotting was similar to that with silver staining where only the O-Ag expression took place. The complementation with the functional waaLOS and waaLPS genes showed inhibi- tion of O-Ag expression and full recovery of OC in both cases. Disruption in the waaLPS gene as a single muta- tion leads to hardly noticeable stimulation of the O-Ag expression. Thereby, the data obtained from silver staining and immunoblotting of DOC-PAGE evidence the participa- tion of WaaL ligase in the LPS phenotype creation. How- ever, strict substrate specificity of the LPS ligase in YeO3 was not detected. Conclusions. Summarizing the obtained data we can conclude that the LPS ligases of YeO3 exhibit rela- xed donor substrate specificity. It has been established that under given conditions the effect of the WaaLOS li- gase is more significant for the OC and OPS ligation on- to lipid A than for the WaaLPS ligase. It is possible that deletion of the waaLOS and waaLPS genes and changes in the OC or O-Ag moieties of LPS enhance the ability of pathogen to evade host defenses. Further work is re- quired to elucidate the biological significance of these different settings. Funding. This work was supported by grants from Center for International Mobility (CIMO), Finland TM-12-8286. 446 SHEVCHENKO J. I. ET AL. YeO3 YeO3 YeO3 YeO3 wt �os �ps �os_�ps 6dA GaNa 6dA 6dA 6dA 6dA 6dA 6dA Glc Glc Glc Glc GlcN GlcN Gal GaNa FuNa Hep2 Hep4Hep3 Hep1 Kdo O-antigen Outer core Inner core Lipid A O-Ag Fig. 3. Silver-stained DOC-PAGE analysis of LPS phenotypes of dif- ferent ligase mutants wt �os �os + os �ps �ps + ps d� d� + os d� + ps Fig. 4. Immunoblot analysis of LPS phenotypes of waaL mutants of YeO3. DOC-PAGE membrane was probed with mAb 2B5 and TomA6 Âïëèâ äåëåö³é ãåí³â ë³ãàç waaL íà ôåíîòèï ë³ïîïîë³ñàõàðèä³â ó áàêòåð³é Yersinia enterocolitica O:3 Þ. ². Øåâ÷åíêî , Â. Ê. Ïîçóð, Ì. Ñêóðíèê Ðåçþìå Ìåòà. Äîñë³äèòè ó÷àñòü ë³ãàç WaaL ó ôîðìóâàíí³ ôåíîòèïó ë³ïî- ïîë³ñàõàðèäó (LPS) ñåðåä áàêòåð³é Y. enterocolitica O:3 (YeO3). Ìåòîäè. Íîêàóòí³ ìóòàíòè ïî ãåíàõ ë³ãàç waaL ñòâîðåíî âíàñë³- äîê îáì³íó àëåëÿìè. Ôåíîòèïè LPS îòðèìàíèõ ìóòàíò³â â³çóàë³- çóâàëè, çàáàðâëþþ÷è ñð³áëîì ãåëü DOC-PAGE, à òàêîæ âèêîðè- ñòîâóâàëè ³ìóíîáëîò ç³ ñïåöèô³÷íèìè ìîíîêëîíàëüíèìè àíòèò³- ëàìè äî êîðó (êîðîâîãî îë³ãîñàõàðèäó, ãåêñàñàõàðèäó, ÎC) òà Î- ïîë³ñàõàðèäó (OPS, O-Ag). Ðåçóëüòàòè. Äåëåö³ÿ ãåíà ë³ãàçè waaLOS ç ãåíîìó áàêòåð³é YeO3 ÷èíèòü ïîì³òíèé âïëèâ íà ë³ãóâàííÿ ÎC ÿê â îäèíî÷íèõ, òàê ³ â ïîäâ³éíèõ ìóòàíòàõ. Ïðîòå ìàí³ïóëÿö³¿ ç ãåíîì ë³ãàçè waaLPS ïðèçâîäÿòü äî ëåäü ïîì³òíî¿ ñòèìóëÿö³¿ óò- âîðåííÿ OPS. Âèñíîâêè. Ë³ãàçè LPS áàêòåð³é YeO3 äåìîíñòðó- þòü íèçüêó ñóáñòðàòíó ñïåöèô³÷í³ñòü. Ó÷àñòü ë³ãàçè WaaLOS ó ôîðìóâàíí³ ïîâíîö³ííî¿ ñòðóêòóðè LPS º ñóòòºâ³øîþ, àí³æ WaaLPS, çà äàíèõ óìîâ. Êëþ÷îâ³ ñëîâà: ë³ãàçè WaaL, LPS, Yersinia enterocolitica, DOC- PAGE. Âëèÿíèå äåëåöèé ãåíîâ ëèãàç waaL íà ôåíîòèï ëèïîïîëèñàõàðèäîâ ó áàêòåðèé Yersinia enterocolitica O:3 Þ. È. Øåâ÷åíêî, Â. Ê. Ïîçóð, Ì. Ñêóðíèê Ðåçþìå Öåëü. Èññëåäîâàòü ó÷àñòèå ëèãàç WaaL â ôîðìèðîâàíèè ôåíîòè- ïà ëèïîïîëèñàõàðèäà (LPS) ñðåäè áàêòåðèé Y. enterocolitica O:3 (YeO3). Ìåòîäû. Íîêàóòíûå ìóòàíòû ïî ãåíàì ëèãàç waaL ñî- çäàíû âñëåäñòâèå îáìåíà àëëåëÿìè. Ôåíîòèïû LPS ïîëó÷åííûõ ìóòàíòîâ âèçóàëèçèðîâàëè, îêðàøèâàÿ ñåðåáðîì ãåëü DOC-PAGE, à òàêæå ñ èñïîëüçîâàíèåì èììóíîáëîòà ñî ñïåöèôè÷åñêèìè ìî- íîêëîíàëüíûìè àíòèòåëàìè ê êîðó (êîðîâîìó îëèãîñàõàðèäó, ãåê- ñàñàõàðèäó, OC) è Î-ïîëèñàõàðèäó (OPS, O-Ag). Ðåçóëüòàòû. Äå- ëåöèÿ ãåíà ëèãàçû waaLOS èç ãåíîìà áàêòåðèé YeO3 îêàçûâàåò çà- ìåòíîå âëèÿíèå íà ëèãèðîâàíèå OC êàê â îäèíî÷íûõ, òàê è äâîé- íûõ ìóòàíòàõ. Îäíàêî ìàíèïóëÿöèè ñ ãåíîì ëèãàç waaLPS ïðèâî- äÿò ê åäâà çàìåòíîé ñòèìóëÿöèè îáðàçîâàíèÿ OPS. Âûâîäû. Ëè- ãàçû LPS áàêòåðèé YeO3 äåìîíñòðèðóþò íèçêóþ ñóáñòðàòíóþ ñïåöèôè÷íîñòü. Ó÷àñòèå ëèãàçû WaaLOS â îáðàçîâàíèè ïîëíîöåí- íîé ñòðóêòóðû LPS ÿâëÿåòñÿ áîëåå ñóùåñòâåííûì, ÷åì WaaLPS, ïðè äàííûõ óñëîâèÿõ. Êëþ÷åâûå ñëîâà: ëèãàçû WaaL, LPS, Yersinia enterocolitica, DOC- PAGE. REFERENCES 1. Ortiz Martinez P, Mylona S, Drake I, Fredriksson-Ahomaa M, Korkeala H, Corry JE. Wide variety of bioserotypes of entero- pathogenic Yersinia in tonsils of English pigs at slaughter. Int J Food Microbiol. 2010;139(1–2):64–9. 2. Skurnik M, Venho R, Toivanen P, al-Hendy A. A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccha- ride outer core biosynthesis. Mol Microbiol. 1995;17(3):575–94. 3. Sabina Y, Rahman A, Ray RC, Montet D. Yersinia enterocoliti- ca: mode of transmission, molecular insights of virulence, and pathogenesis of infection. J Pathog. 2011;2011:429069. 4. Pinta E, Li Z, Batzilla J, et al. Identification of three oligo-poly- saccharide-specific ligases in Yersinia enterocolitica. Mol Mic- robiol. 2012;83(1):125–36. 5. Biedzka-Sarek M, Venho R, Skurnik M. Role of YadA, Ail, and li- popolysaccharide in serumresistance of Yersinia enterocolitica serotype O:3. Infect Immun. 2005;73(4):2232–44. 6. Skurnik M. Lack of correlation between the presence of plas- mids and fimbriae in Yersinia enterocolitica and Yersinia pseu- dotuberculosis. J Appl Bacteriol. 1984;56(3):355–63. 7. Babic A, Guerout AM, Mazel D. Construction of an improved RP4 (RK2)-based conjugative system. Res Microbiol. 2008;159(7– 8):545–9. 8. Wilson KJ, Sessitsch A, Corbo JC, Giller KE, Akkermans AD, Jefferson RA. beta-Glucuronidase (GUS) transposons for ecolo- gical and genetic studies of rhizobia and other gram-negative bacteria. Microbiology. 1995;141 (Pt 7):1691–705. Received 11.07.14 447 THE EFFECT OF waaL GENES DELETION FROM Yersinia enterocolitica O:3 GENOME

The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype

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