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Characterization of a Transposon Tn5-Generated Mutant of Yersinia pestis Defective in Lipooligosaccharide Biosynthesis

R. Z. Shaikhutdinova1, S. A. Ivanov1, S. V. Dentovskaya1,a,b*, G. M. Titareva1, and Yu. A. Knirel2,c,d

1State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Moscow Region, Russia

2Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia

* To whom correspondence should be addressed.

Received September 7, 2018; Revised October 25, 2018; Accepted October 25, 2018
To identify Yersinia pestis genes involved in the microbe’s resistance to cationic antimicrobial peptides, the strategy of random transposon mutagenesis with a Tn5 minitransposon was used, and the library was screened for detecting polymyxin B (PMB) susceptible mutants. The mutation responsible for PMB-sensitive phenotype and the lipopolysaccharide (LPS) structure were characterized for the Y. pestis strain KM218-A3. In this strain the mini-Tn5 was located in an open reading frame with the product homologous to the E. coli protein GmhB (82% identity) functioning as d-glycero-d-manno-heptose-1,7-diphosphate phosphatase. ESI FT ICR mass spectrometry of anions was used to study the structure of the unmodified LPS of Y. pestis KM218-A3, and molecules were revealed with the full-size LPS core or with two types of an incomplete core: consisting of Kdo-Kdo or Ko-Kdo disaccharides and Hep-(Kdo)-Kdo or Hep-(Ko)-Kdo trisaccharides. The performed complementation confirmed that the defect in the biological properties of the mutant strain was caused by inactivation of the gmhB gene. These findings indicated that the gmhB gene product of Y. pestis is essential for production of wild-type LPS resistant to antimicrobial peptides and serum.
KEY WORDS: Yersinia pestis, lipopolysaccharide, heptose biosynthesis, serum resistance, antibiotic resistance

DOI: 10.1134/S0006297919040072