The Glycine Lipids of Bacteroides thetaiotaomicron Are Important for Fitness during Growth In Vivo and In Vitro

Abstract

Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the N-acylation of the amino acid, and this is followed by subsequent O-acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced in Bacteroides thetaiotaomicron. We, and others, have previously reported the identification of a gene, named glsB in this study, that encodes an N-acyltransferase activity responsible for the production of a monoacylated glycine called N-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of the Bacteroidales genomes sequenced so far, the glsB gene is located immediately downstream from a gene, named glsA, that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression of glsB and glsA results in the production of GL in Escherichia coli. We constructed a deletion mutant of the glsB gene in B. thetaiotaomicron, and we confirm that glsB is required for the production of GL in B. thetaiotaomicron. Moreover, we show that glsB is important for the ability of B. thetaiotaomicron to adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacterium B. thetaiotaomicron. IMPORTANCE The gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from the Bacteroidales, an order that includes Bacteroides and Prevotella. In this study, we have identified an acylated amino acid, called glycine lipid, produced by Bacteroides thetaiotaomicron, a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation of B. thetaiotaomicron to a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut by B. thetaiotaomicron. This work identifies glycine lipids as an important fitness determinant in B. thetaiotaomicron and therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.

Elucidation of Gut Microbiota-Associated Lipids Using LC-MS/MS and 16S rRNA Sequence Analyses

“…Furthermore, our results showed that the bacteria correlating with AAHFA metabolites were highly correlated with NAGly and NAGlySer molecules, which also have the structural backbone of fatty acid esters of hydroxy fatty acyls (lipid classes D-1 and D-2 in Figure 4). Importantly, NAGly molecules are biosynthesized in Bacteroides by N-acyltransferase (glsB), followed by the O-acyltransferase (glsA) enzymatic reaction (Lynch et al, 2019). Thus, it indicates that these bacteria would have an esterifying enzyme for a-hydroxy fatty acids to form AAHFAs.…”

Characterization of a novel AraC/XylS-regulated family of N-acyltransferases in pathogens of the order Enterobacterales

“…Myristoylated proteins have a myristate linked to Gly-2 via an amide, whereas palmitoylated proteins contain palmitate linked to the N-terminal (N-acylation) or the sulfhydryl group (S-acylation) of cysteine [ 12 – 14 ]. The Bacteroidales produce an N-acylated derivative of glycine, which is another subclass of the N-fatty acyl amino acids [ 15 ].…”

Mapping the myristoylome through a complete understanding of protein myristoylation biochemistry

“…It is also interesting to highlight that glycine lipidation with C14, C16, and C18 derivatives and the associated acyl transferase GlsB occurs in Bacteroides, a Gram-negative, obligate anaerobic bacterium [330,331]. Mono glycine lipids are membrane components important for growth [331].…”

“…It is also interesting to highlight that glycine lipidation with C14, C16, and C18 derivatives and the associated acyl transferase GlsB occurs in Bacteroides, a Gram-negative, obligate anaerobic bacterium [330,331]. Mono glycine lipids are membrane components important for growth [331]. However, the bacterial transferases appear to be more closely related to the lysophospholipid acyltransferase superfamily (pfam13444/cl17185), which include various lipid acceptors different from polypeptides and including polysaccharides such as in lipid A [332].…”