https://microbiolj.org.ua/en/archiv/2022-tom-84/6-nov-dec-tom-84/2022-84-6-02

Q4 Biochemistry, Genetics and Molecular Biology
T. V. Bulyhina, L. Pasichnyk, K. Garkava
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引用次数: 0

摘要

细菌性湿木,细菌性水肿,或细菌性粘液是一种常见的疾病,由小叶莲子引起,影响许多针叶树和落叶树的中心核心。该物种的代表已从表现出该病症状的各种树木中分离出来,也从水中分离出来,较不常见的是从临床样本中分离出来。病原菌的识别过程和细菌细胞对植物抗菌物质的保护机制是其发病机制的重要方面。已知脂多糖(LPS)在这些过程中起着积极的作用。它们提供外膜的屏障功能,帮助保护细菌免受植物抗微生物化合物的侵害,并使细菌附着在植物细胞上。因此,本研究的目的是研究不同方法获得的油压小榄F9a1脂多糖的化学成分和功能生物学特性的特点。方法。采用苯酚-水法(LPS I)、0.85% NaCl溶液提取法(LPS II)和不溶于NaCl溶液的苯酚-水提取法(LPS III)从干菌群中分离LPS,碳水化合物采用Dubois法分析,核酸采用Spirin法分析,蛋白质含量采用Lowry法分析,2-酮-3-脱氧辛酸(KDO)法分析。采用Agilent 6890N/5973惰性质谱联用系统对LPS制剂中的单糖和脂肪酸进行鉴定。按照生物伦理学的原则,在家兔身上进行了LPS的热原性实验。采用Ouchterlony法进行血清学研究。结果。L. nimipressuralis f9sp1的LPS II具有相对产量低(2.12%)、碳水化合物含量低(9.16%)和核酸含量低(3.7%)、蛋白质含量高(26.44%)的特点,而LPS I和LPS III的制备具有产量高、碳水化合物含量较高(分别为46.68%和38.4%)、蛋白质含量不高(高达6.72%)和核酸含量低(高达4.06%)的特点。所有LPS均含有高达0.27%的KDO, KDO是革兰氏阴性菌LPS的特异性成分。单糖组成表明,所研究的nimipressuralis菌株的LPS是异质性的。同时,在所有被试菌株的LPS中记录了焦糖、半乳糖和葡萄糖等单糖。脂多糖的脂肪酸组成由含有12至18个碳原子的脂肪酸组成。Нydroxylated,饱和酸和单不饱和酸。在LPS I和LPS III中,优势脂肪酸为14:0 (3-OH),这是整个肠杆菌科的一种标记。除了标记酸外,在LPS III中也以16:1酸为主,而在LPS II中,16:1(32.7%)和16:0(22.6%)脂肪酸占主导地位。研究结果表明,脂多糖溶液具有热原性。血清学研究表明,被测LPS在同源系统中表现出抗原活性。nimipressuralis F9a1的抗血清与LPS菌株IMV 8791、LGK1和L14b发生反应,这可能表明存在共同的抗原决定因素,并且这些菌株属于同一血清群。结论。L. nimipressuralis F9a1的脂多糖在单糖和脂肪酸组成上存在异质性,这可能与采用不同的分离方法有关。水-苯酚法比氯化钠法更适合从L. nimipressuralis细胞中分离LPS,因为氯化钠法提取率很低,且多糖被蛋白质污染严重。同时,分离方法不影响所研究的LPS的血清学活性。在这些生物功能研究中获得的结果有助于该物种的生物学特性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
https://microbiolj.org.ua/en/archiv/2022-tom-84/6-nov-dec-tom-84/2022-84-6-02
Bacterial wetwood, bacterial dropsy, or bacterial slime is a common disease caused by Lelliottia nimipressuralis, which affects the central core of many conifers and deciduous trees. Representatives of this species have been isolated from a variety of trees showing symptoms of the disease, as well as from water and, less commonly, from clinical samples. Important aspects of pathogenesis is the process of pathogen recognition and the protection mechanisms of bacterial cells from plant’s antimicrobial substances. It is known that lipopolysaccharides (LPS) take an active part in these processes. They provide the barrier function of the outer membrane, helping to protect bacteria from plant antimicrobial compounds, and the attachment of bacteria to plant cells. Therefore, the aim of the work was to study the peculiarities of the chemical composition and functional and biological characteristics of Lelliottia nimipressuralis F9a1 LPS obtained by different methods. Methods. LPS was isolated from dry bacterial mass by phenol-water method (LPS I), extraction method with 0.85% NaCl solution (LPS II), and phenol-water extraction of LPS insoluble in NaCl solution (LPS III). The carbohydrates were analyzed by Dubois method, nucleic acids ‒ by Spirin, protein content ‒ by Lowry and 2-keto-3-deoxyoctonic acid (KDO) ‒ by Osborn. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromato-mass spectrometry system. The pyrogenicity of LPS was tested keeping the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. Results. LPS II of L. nimipressuralis F9а1 was characterized by low relative yield (2.12%), low content of carbohydrates (9.16%) and nucleic acids (3.7%), and high protein content (26.44%), while the studied preparations of LPS I and LPS III were characterized by a high yield, a rather high content of carbohydrates (46.68 and 38.4%, respectively), an insignificant amount of protein (up to 6.72%) and nucleic acids (up to 4.06%). All LPSs contained up to 0.27% KDO, which is a specific component of the LPS of gramnegative bacteria. The monosaccharide composition indicates that the LPS of the studied L. nimipressuralis strains turned out to be heterogeneous. At the same time, such monosaccharides as fucose, galactose, and glucose were recorded in the LPS of all tested strains. The fatty acid composition of LPS was represented by the presence of fatty acids containing from 12 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids were found. In LPS I and LPS III, the dominant fatty acid was 14:0 (3-OH), which is a kind of marker for the entire family of Enterobacteriaceae. In addition to the marker acid, a 16:1 acid was also predominant in LPS III, whereas in LPS II, 16:1 (32.7%) and 16:0 (22.6%) fatty acids dominated. The pyrogenic effect of L. nimipressuralis LPS studied showed that LPS solutions are pyrogenic. The serological studies showed that tested LPS in homologous systems exhibits antigenic activity. Antisera to L. nimipressuralis F9a1 react with LPS strains IMV 8791, LGK1, and L14b, which may indicate the presence of common antigenic determinants and belonging of these strains to the same serogroup. Conclusions. The LPSs of L. nimipressuralis F9a1 were heterogeneous in both monosaccharide and fatty acid composition, which is explained by the use of different methods for their isolation. To isolate LPS from L. nimipressuralis cells, the water-phenol method is better than sodium chloride extraction since with using the latter, the LPS yield is very low and very contaminated with proteins. At the same time, the isolation method does not affect the serological activity of the studied LPS. The results received during these biological-functional studies of L. nimipressuralis LPS contribute to the biological characteristics of this species.
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Mikrobiolohichnyi zhurnal
Mikrobiolohichnyi zhurnal Medicine-Microbiology (medical)
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