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Peptidoglycan compositional analysis of Enterococcus faecalis biofilm by stable isotope labeling by amino acids in bacterial culture

机译：细菌培养中氨基酸稳定同位素标记粪肠球菌生物膜的肽聚糖组成分析

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Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into bacteria’s cellular functions and responses to external stimuli, but this analysis remains challenging due to various chemical modifications to PG-repeat subunits. We characterized changes to the PG composition in E. faecalis grown as planktonic bacteria and biofilm by developing “Stable Isotope Labeling by Amino Acids in Bacterial Culture” (SILAB) optimized for bacterial cultures with incomplete amino acid labeling. This comparative analysis by mass spectrometry was carried out by labeling E. faecalis in biofilm with heavy-Lys (L-[13C6, 2D9, 15N2]Lys) and planktonic bacteria with natural abundance L-Lys, then mixing the equal amount of bacteria from each condition and carrying out cell-wall isolation and mutanolysin digestion necessary for liquid chromatography-mass spectrometry. An analytical method was developed to determine muropeptide abundances using correction factors to compensate for incomplete heavy-Lys isotopic enrichment (98.33% ± 0.05%) and incorporation (83.23% ± 1.16%). SILAB analysis of 47 pairs of PG fragment ions from isolated cell walls of planktonic and biofilm samples were selected for analysis. We found that the PG in biofilm showed increased PG cross-linking, increased N-deacetylation of GlcNAc, decreased O-acetylation of MurNAc, and increased stem modifications by D,D- and L,D-carboxypeptidases.
机译：肽聚糖（PG）是粪肠球菌中细胞壁的主要成分。 PG成分的准确分析可提供细菌细胞功能和对外部刺激反应的关键见解，但由于对PG重复亚基进行了多种化学修饰，因此该分析仍然具有挑战性。通过开发“细菌培养中氨基酸的稳定同位素标记”（SILAB），我们优化了粪便中作为浮游细菌和生物膜生长的粪肠球菌PG组成的变化，该方法针对氨基酸标记不完整的细菌培养进行了优化。质谱法的这种比较分析是通过在生物膜中用重Lys（L-[ 13 C6， 2 D9， 15 N2] Lys）和具有自然丰度L-Lys的浮游细菌，然后将每种条件下等量的细菌混合，然后进行液相色谱-质谱分析所必需的细胞壁分离和变溶酶消化。开发了一种分析方法，可使用校正因子补偿不完整的重Lys同位素富集（98.33％±0.05％）和掺入（83.23％±1.16％）来确定多肽丰度。从浮游生物样品和生物膜样品的分离细胞壁中选择47对PG碎片离子进行SILAB分析，以进行分析。我们发现生物膜中的PG显示增加PG交联，增加GlcNAc的N-去乙酰化，降低MurNAc的O-乙酰化，并增加D，D-和L，D-羧肽酶对茎的修饰。

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