Wahlig Taylor A, Stanton Eliot, Godfrey Jared J, Stasic Andrew J, Wong Amy C L, Kaspar Charles W
Department of Bacteriology, University of Wisconsin, Madison, WI, United States.
U. S. Food and Drug Administration, Center for Biologics Evaluation and Research, Washington, DC, United States.
Front Microbiol. 2021 Jun 2;12:671453. doi: 10.3389/fmicb.2021.671453. eCollection 2021.
The outer membrane of plays an important role in combating stress encountered in the environment and hosts. The transport and insertion of lipopolysaccharides (LPS) into the outer membrane involves lipopolysaccharide transport proteins (LptA-F) and mutations in the genes encoding for these proteins are often lethal or result in the transport of atypical LPS that can alter stress tolerance in bacteria. During studies of heterogeneity in bile salts tolerance, serovar Typhimurium E40 was segregated into bile salts tolerant and sensitive cells by screening for growth in TSB with 10% bile salts. An isolate (E40V) with a bile salts MIC >20% was selected for further characterization. Whole-genome sequencing of E40 and E40V using Illumina and PacBio SMRT technologies revealed a non-synonymous single nucleotide polymorphism (SNP) in . Leucine at residue 26 in E40 was substituted with proline in E40V. In addition to growth in the presence of 10% bile salts, E40V was susceptible to novobiocin while E40 was not. Transcriptional analysis of E40 and E40V, in the absence of bile salts, revealed significantly greater ( < 0.05) levels of transcript in three genes in E40V; (encoding for an extracellular polymeric substance production protein), (encoding for a putative stress response protein), and an uncharacterized gene annotated as an acid shock protein precursor (ASPP). No transcripts of genes were present at a greater level in E40 compared to E40V. Corresponding with the greater level of these transcripts, E40V had greater survival at pH 3.35 and staining of Calcofluor-binding polysaccharide (CBPS). To confirm the SNP in was associated with these phenotypes, strain E40E was engineered from E40 to encode for the variant form of LptG (L26P). E40E exhibited the same differences in gene transcripts and phenotypes as E40V, including susceptibility to novobiocin, confirming the SNP was responsible for these differences.
[细菌名称]的外膜在应对环境和宿主中遇到的压力方面发挥着重要作用。脂多糖（LPS）向外膜的转运和插入涉及脂多糖转运蛋白（LptA - F），编码这些蛋白的基因突变通常是致死性的，或者会导致非典型LPS的转运，从而改变细菌的应激耐受性。在对胆汁盐耐受性异质性的研究中，通过在含有10%胆汁盐的胰酪大豆胨肉汤（TSB）中筛选生长情况，将鼠伤寒沙门氏菌血清型Typhimurium E40分离为胆汁盐耐受型和敏感型细胞。选择一株胆汁盐最低抑菌浓度（MIC）>20%的分离株（E40V）进行进一步表征。使用Illumina和PacBio SMRT技术对E40和E40V进行全基因组测序，发现在[基因名称]中有一个非同义单核苷酸多态性（SNP）。E40中第26位残基的亮氨酸在E40V中被脯氨酸取代。除了在10%胆汁盐存在下生长外，E40V对新生霉素敏感，而E40则不敏感。在无胆汁盐的情况下对E40和E40V进行转录分析，结果显示E40V中三个基因的转录水平显著更高（P < 0.05）；[基因1名称]（编码一种胞外聚合物产生蛋白）、[基因2名称]（编码一种假定的应激反应蛋白），以及一个注释为酸休克蛋白前体（ASPP）的未表征基因。与E40V相比，E40中这些基因的转录本水平没有更高。与这些转录本水平较高相对应，E40V在pH 3.35时具有更高的存活率以及荧光增白剂结合多糖（CBPS）染色。为了确认[基因名称]中的SNP与这些表型相关，从E40构建了菌株E40E，以编码LptG的变体形式（L26P）。E40E在基因转录本和表型上表现出与E40V相同的差异，包括对新生霉素的敏感性，证实该SNP导致了这些差异。
