Structural insights into CodY activation and DNA recognition.

Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage.

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.

Modeling microbial ethanol production by S. aureus, K. pneumoniae, and E. faecalis under aerobic/anaerobic conditions - applicability to laboratory cultures and real postmortem cases.

A quite intriguing subject being intensively researched in the forensic toxicology field is the source of postmortem determined blood ethanol concentration: antemortem ingestion or postmortem microbial production. Our previous research on microbial ethanol production has reported a quantitative relationship between the ethanol and the higher alcohols and 1-butanol produced by Escherichia coli, Clostridium perfrigens, and Clostridium sporogenes. In this contribution, we continue our research reporting on the following: (i) the patterns of ethanol, higher alcohols, and 1-butanol production by the microbes Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (all being aerobic/facultative anaerobic species, common corpse's colonizers, and ethanol producers), under controlled laboratory conditions, (ii) the mathematical modeling, with simple mathematical equations, of the correlation between ethanol concentration and the other studied alcohols' concentrations, by performing multiple linear regression analysis of the results, and (iii) the applicability of the constructed models in microbial cultures developed under different temperature than that used to build the models, in denatured blood cultures and in real postmortem cases. The aforementioned alcohols were proved to be all indicators of ethanol production, both in qualitative and quantitative terms. 1-Propanol was the most significant alcohol in modeling microbial ethanol production, followed by methyl-butanol. The K. pneumoniae's models achieved the best scoring in applicability (E < 40%) compared to the S. aureus and E. faecalis models, both at laboratory microbial cultures at 37 °C and real postmortem cases. Overall, a noteworthy accuracy in estimating the microbial ethanol in cultures and autopsy blood is achieved by the employed simple linear models.

Isolation and Identification of Dominant Bacteria from Raw Donkey Milk Produced in a Region of Morocco by QIIME 2 and Evaluation of Their Antibacterial Activity.

Recently, the interest in donkey milk has increased considerably because it proved high nutritive and functional values of their ingredients. Its chemical composition is widely studied, but its microbiota, especially lactic acid bacteria, remains less studied. This study focuses on analyzing, isolating, and identifying lactic acid bacteria and evaluating their capacity to produce biomolecules with antibacterial activity. Among 44 strains identified, 43 are Gram-positive, and most are catalase-negative and cocci-shaped. Five strains were selected to evaluate their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. Different induction methods allowed to amplify the antibacterial effects against these pathogenic strains.

Regulation of growth, intestinal microflora composition and expression of immune-related genes by dietary supplementation of Streptococcus faecalis in blunt snout bream (Megalobrama amblycephala).

A 10-week feeding trial was performed to investigate the effects of Streptococcus faecalis on the growth, intestinal microflora composition and expression of immune-related genes of blunt snout bream (Megalobrama amblycephala). Fish (46.32 ± 0.09 g) were fed four experimental diets containing 0 cfu/g (SF0, control), 1 × 105 cfu/g (SF1), 1 × 106 cfu/g (SF2) and 1 × 107 cfu/g (SF3) of S. faecalis, respectively. Results showed that daily growth index (DGI), feed efficiency ratio (FER), plasma glucose level, plasma contents of total protein and albumin as well as intestinal serous layer (SL), muscular layer (ML), submucous layer (SML), villi thickness (VT) and lamina propria (LP) were all no significant difference among all the treatments, whereas their (except plasma albumin content and intestinal ML) relatively high values were found in the SF2 group. Meanwhile, the intake of the SF2 diets significantly increased plasma globulin content and intestinal digestive enzymes activities, the opposite was true for the activities of plasma aspartate aminotransferase (AST) and alanine transaminase (ALT). In addition, the analysis of the intestinal microbiota showed that fish fed the SF2 diet have the highest values of intestinal alpha diversity and intestinal abundances of Actinobacteria, Chlamydiae, Firmicutes, Planctomycetes, Verrucomicrobia, Clostridium and Synechococcus, while the opposite was true for intestinal abundances of Acinetobacter, Anoxybacillus, Flavobacterium, Planctomyces, Plesiomonas, Pseudomonas, Staphylococcus and Clostridium perfringens. At the molecular level, the expression levels of tumour necrosis factor α (TNF α), interleukin 1ß (IL 1ß) and heat shock proteins 7 (HSP 70) in head kidney and spleen were all decreased significantly with the increasing S. faecalis levels up to 1 × 106 cfu/g, and then they were increased with further increasing S. faecalis levels. Overall, dietary supplementation of S. faecalis at 1 × 106 cfu/g could improve the intestinal health and innate immunity of blunt snout bream.

Effects of single or conjoint administration of lactic acid bacteria as potential probiotics on growth, immune response and disease resistance of snakehead fish (Channa argus).

Lactic acid bacteria (LAB) has been documented to promoting growth, enhancing immunity and disease resistance. In this study, we aimed to evaluate the single or conjoint effects of Lactococcus lactis L19 (Genbank: MT102745.1) and Enterococcus faecalis W24 (Genbank: MT102746.1) isolated from the intestine of Channa argus (C. argus) on growth performance, immune response and disease resistance of C. argus. A total of 720 apparently healthy C. argus (9.50 ± 0.03 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with L. lactis (L19), E. faecalis (W24), and L. lactis L19 + E. faecalis W24 (L + W) at 1.0 × 108 cfu/g basal diet for 56 days. After feeding, the final body weight (FBW), weight gain (WG), feed efficiency ratio (FER), specific growth rate (SGR) and protein efficiency ratio (PER) had significantly increased (p < 0.05), especially with L19. The results indicated that single or conjoint administration of LAB as potential probiotics can induce high levels of IgM, ACP, AKP, LZM, C3 and C4 activity in serum, which may effectively induce humoral immunity, and L19 induce even higher levels. Meanwhile, when compared to CK group, the results of qPCR showed that LAB administration significantly up-regulated (p < 0.05) the expression of IL-1ß, IL-6, IL-10, TNF-α, IFN-γ, HSP70, HSP90, TGF-ß in the spleen, head kidney, gill, liver and intestine of C. argus. After challenge with Aeromonas veronii, the survival rates in all LAB-fed groups were significantly higher (p < 0.05) than that of the CK group, and the L19 group showed the highest (63.3%) disease resistance. Our data indicated that L. lactis L19 and E. faecalis W24, as a feed additive at 1.0 × 108 cfu/g feed, could promote growth performance, enhance immune response and disease resistance of C. argus, with greatest effects in fish fed L. lactis L19 for 56 days. Hence, these LAB additives could be used as promising probiotics for C. argus. L19 was more effective than W24 or the mixture of the two for promoting growth performance, enhancing immune response and disease resistance of C. argus.

Reisolation and Configurational Reinvestigation of Cottoquinazolines E-G from an Arthropod-Derived Strain of the Fungus Neosartorya fischeri.

The reported fumiquinazoline-related alkaloids cottoquinazolines E-G (1-3) were reisolated from solid cultures of the fungus Neosartorya fischeri, which was isolated from the medicinal arthropod Cryptotympana atrata. The unresolved issues regarding the absolute configurations (for cottoquinazolines E and F) prompted a reinvestigation of the configurations for all three compounds, as enabled by extensive spectroscopic methods, comparisons of experimental electronic circular dichroism data, and X-ray crystallography. In addition, cottoquinazoline F (2) showed significant antibacterial activity against ESBL-producing Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis with MIC values of 8, 32, 32, and 16 µg/mL, respectively.

Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA.

The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ:RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5α. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria.

Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. To capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two-photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG not only can monitor the movement of small molecules across membrane leaflets but also is sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.

Colorimetric and Electrochemical Detection of Escherichia coli and Antibiotic Resistance Based on a p-Benzoquinone-Mediated Bioassay.

The facile and economical identification of pathogenic bacteria, especially their antibiotic-resistance, is crucial in the realm of human health and safety. The presence of Escherichia coli ( E. coli) is considered as an indicator of water contamination and is closely related to human health. Herein, inspired by the biocatalysis of bacterial surfaces, we developed a simple and cost-effective colorimetric- and electrochemical-based bioassay that is capable of analyzing both the presence of E. coli and its relative level of antibiotic resistance. In this approach, p-benzoquinone is used as a redox mediator to monitor the bacterial concentration and specifically distinguish E. coli from four other common clinical bacteria, namely, Staphylococcus aureus ( S. aureus), Enterococcus faecalis ( E. faecalis), Salmonella pullorum ( S. pullorum), and Streptococcus mutans ( S. mutans). A visible color change, captured with a smartphone using a "light box", without relying on any complex instruments, can reflect the concentration of bacteria. The accurate quantification of E. coli was investigated with an electrochemical system in the concentration ranges of 1.0 × 103 to 1.0 × 109 CFU/mL. We further demonstrated the capability of the presented biosensor in identifying drug-resistant bacteria with two artificially induced antibiotic-resistant bacteria. Therefore, the presented bioassay is not only capable of detecting E. coli with high sensitivity and specificity but also provides a rapid solution to evaluate E. coli antibiotic resistance.

Lactic acid bacteria, Enterococcus faecalis Y17 and Pediococcus pentosaceus G11, improved growth performance, and immunity of mud crab (Scylla paramamosain).

Mud crabs (Scylla paramamosain), a commercially important cultured species in the southeastern region of China, is usually infected by Vibriosis or parasites, causing great economic losses in cultured farms. Previous studies have demonstrated that probiotics benefited in enhancing the immune response against invading pathogens in aquatic animals. In this study, the effects of dietary administration of lactic acid bacteria (LAB) (Enterococcus faecalis Y17 and Pediococcus pentosaceus G11) on growth performance and immune responses of mud crab were assessed. Both strains (Y17 and G11) showed an inhibitory activity against bacterial pathogens (Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, and ß Streptococcus), and a wide pH tolerance range of 2-10. In vivo, mud crabs were fed a control diet and experimental diets supplemented with 109 cfu g-1 diet either Y17 or G11 for 6 weeks before subjecting to a challenge test with V. parahaemolyticus for 12 h. The probiotic-supplemented diets had significant effects on weight gain and specific growth rate during the feeding trial. Increased serum enzyme activities of phenoloxidase, lysozyme, and SOD were observed in the hemolymph of mud crab in Y17 and G11-supplemented groups compared to that in the controls (P < 0.01). The significantly up-regulated expression of gene CAT, LYS, proPO, and SOD could be seen in hepatopancreas in G11-supplemented groups. After the pathogenicity test, the survival rate of Y17 + and G11 + V. parahaemolyticus groups was 66.67% and 80.00%, respectively, compared with 53.33% for the control groups. Taken together, dietary supplementation of Y17 and G11 strains were beneficial in mud crab, which could increase growth performance, modulate immune system and protect the host against V. parahaemolyticus infection.

Pheromone peptide cOB1 from native Enterococcus faecalis forms amyloid-like structures: A new paradigm for peptide pheromones.

Pheromone peptides are an important component of bacterial quorum-sensing system. The pheromone peptide cOB1 (VAVLVLGA) of native commensal Enterococcus faecalis has also been identified as an antimicrobial peptide (AMP) and reported to kill the prototype clinical isolate strain of E. faecalis V583. In this study, the pheromone peptide cOB1 has shown to form amyloid-like structures, a characteristic which is never reported for a pheromone peptide so far. With in silico analysis, the peptide was predicted to be highly amyloidogenic. Further, under experimental conditions, cOB1 formed aggregates displaying characteristics of amyloid structures such as bathochromic shift in Congo red absorbance, enhancement in thioflavin T fluorescence, and fibrillar morphology under transmission electron microscopy. This novel property of pheromone peptide cOB1 may have some direct effects on the binding of the pheromone to the receptor cells and subsequent conjugative transfer, making this observation more important for the therapeutics, dealing with the generation of virulent and multidrug-resistant pathogenic strains.

Effects of Enterococcus faecalis on egg production, egg quality and caecal microbiota of hens during the late laying period.

This study was conducted to determine the effects of diet supplementation of laying hens with Enterococcus faecalis (EF) on egg production, egg quality and caecal microbiota. A total of 360 Hy-Line Brown laying hens (72 weeks old) were divided into three groups with four replicates of 30 birds each. The laying hens were fed with the basal diet (Control), the basal diet + 3.75 · 108 cfu EF/kg (Group I) or the basal diet + 7.5 · 108 cfu EF/kg (Group II). The experiment lasted for 45 d. Eggs and caecal samples were collected at the end of the experiment. Results showed that dietary supplementation with EF did not affect the average daily egg weight, cracked egg rate, mortality and egg quality. However, EF supplementation caused a significantly increased laying rate and decreased feed/egg ratio (p < 0.05). The differences in caecal microbiota between Group II and the Control were significant. The relative abundance of Verrucomicrobia and Cyanobacteria at the phylum level, Rikenellaceae, Christensenellaceae and Veillonellaceae at the family level, and the Faecalibacterium, Christensenellaceae R-7 group and Eubacterium coprostanoligenes group at the genus level changed significantly in Group II compared with the Control (p < 0.05). In conclusion, the tested dietary supplementations with EF improved product performance and affected the caecal microbial community structure of laying hens during the late laying period.

[Evaluation of anti-candida activity of metabolites of enterococcal clinical isolates.]

When studying the effect of the metabolic products of clinical isolates of enterococci on the viability of Candida albicans, it was found that metabolites of all tested strains of Enterococcus faecium, E. faecalis had a fungistatic effect. At the same time a reliable fungicidal effect is a strain-specific feature. It is better to use the method of delayed antagonism on double-layer agar to assess the antifungal effect of enterococcal metabolism products.

Peptidoglycan Compositional Analysis of Enterococcus faecalis Biofilm by Stable Isotope Labeling by Amino Acids in a Bacterial Culture.

Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into the bacterium's cellular functions and responses to external stimuli, but this analysis remains challenging because of various chemical modifications to PG-repeat subunits. We characterized changes to the PG composition of 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 performed by labeling E. faecalis in biofilm with heavy Lys (l-[13C6,2D9,15N2]Lys) and planktonic bacteria with natural abundance l-Lys, then mixing equal amounts of bacteria from each condition, and performing cell wall isolation and mutanolysin digestion necessary for liquid chromatography and 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%). Forty-seven pairs of PG fragment ions from isolated cell walls of planktonic and biofilm samples were selected for SILAB analysis. We found that the PG in biofilm showed an increased level of PG cross-linking, an increased level of N-deacetylation of GlcNAc, a decreased level of O-acetylation of MurNAc, and an increased number of stem modifications by d,d- and l,d-carboxypeptidases.

The evaluation of E. faecalis colonies dissolution ability of sodium hypochlorite in microenvironment by a novel device.

Enterococcus faecalis(E. faecalis) is a common microorganism could be isolated from the infected canals, especially in the case of refractory apical periodontitis. Due to its ability to invade the dentinal tubules and highly resistant to antimicrobial strategies, the thorough debridement of E.faecalis is hard to achieve. And that may be one of the reasons to cause reinfection and therapeutic failure. According to the anatomy of dentinal tubules published before and the results of our team previous work, we designed six types of microtubes with different sizes. By using the method of centrifugation and incubation, a standard infected model mimicking dentinal tubules was established. Sodium hypochlorite (NaClO) is the most popular irrigant applied in root canal treatment. We used three different concentrations with four distinct irrigation duration to observe the antibacterial process of E. faecalis colonies within microtubes dynamically. We concluded that the role of NaClO in the microtubes is concentration dependent and duration dependent. And the interpretation of the results has a certain reference value for clinicians.

Cryo-EM structure of the tetracycline resistance protein TetM in complex with a translating ribosome at 3.9-Å resolution.

Ribosome protection proteins (RPPs) confer resistance to tetracycline by binding to the ribosome and chasing the drug from its binding site. Current models for RPP action are derived from 7.2- to 16-Å resolution structures of RPPs bound to vacant or nontranslating ribosomes. Here we present a cryo-electron microscopy reconstruction of the RPP TetM in complex with a translating ribosome at 3.9-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM with a unique splayed conformation of nucleotides A1492 and A1493 at the decoding center of the small subunit. The resolution enables us to unambiguously model the side chains of the amino acid residues comprising loop III in domain IV of TetM, revealing that the tyrosine residues Y506 and Y507 are not responsible for drug-release as suggested previously but rather for intrafactor contacts that appear to stabilize the conformation of loop III. Instead, Pro509 at the tip of loop III is located directly within the tetracycline binding site where it interacts with nucleotide C1054 of the 16S rRNA, such that RPP action uses Pro509, rather than Y506/Y507, to directly dislodge and release tetracycline from the ribosome.

Surface-Enhanced Raman Spectroscopy for the Detection of a Metabolic Product in the Headspace Above Live Bacterial Cultures.

In situ surface-enhanced Raman spectra of the headspace above cultures of six bacterial species showed strong characteristic bands from chemisorbed methyl sulfide. This marker compound is created by dissociation of dimethyl disulfide (DMDS), a fermentative metabolite of bacteria, on the surface of the enhancing Au or Ag nanoparticle films. Kinetic binding plots of media spiked with DMDS and of live cultures showed that the Au-based substrates were more suitable for the rapid detection of bacteria than Ag-based substrates. For E. coli DH5α, the sensitivity limit for headspace SERS detection was 1.5×107  CFU mL-1 , which corresponded to detection 15 min after inoculation of the growth medium. Since the metabolites are only produced by viable bacteria, antibiotic (gentamicin) treatment stopped the normal signal growth of the marker peak. This work is a promising step towards rapid bedside detection of bacterial infections and rapid screening of antibiotics against bacteria.

Identification of the Lyso-Form N-Acyl Intramolecular Transferase in Low-GC Firmicutes.

Bacterial lipoproteins are embedded in the cell membrane of both Gram-positive and Gram-negative bacteria, where they serve numerous functions central to cell envelope physiology. Lipoproteins are tethered to the membrane by an N-acyl-S-(mono/di)-acyl-glyceryl-cysteine anchor that is variously acylated depending on the genus. In several low-GC, Gram-positive firmicutes, a monoacyl-glyceryl-cysteine with an N-terminal fatty acid (known as the lyso form) has been reported, though how it is formed is unknown. Here, through an intergenic complementation rescue assay in Escherichia coli, we report the identification of a common orthologous transmembrane protein in both Enterococcus faecalis and Bacillus cereus that is capable of forming lyso-form lipoproteins. When deleted from the native host, lipoproteins remain diacylated with a free N terminus, as maturation to the N-acylated lyso form is abolished. Evidence is presented suggesting that the previously unknown gene product functions through a novel intramolecular transacylation mechanism, transferring a fatty acid from the diacylglycerol moiety to the α-amino group of the lipidated cysteine. As such, the discovered gene has been named lipoprotein intramolecular transacylase (lit), to differentiate it from the gene for the intermolecular N-acyltransferase (lnt) involved in triacyl lipoprotein biosynthesis in Gram-negative organisms.IMPORTANCE This study identifies a new enzyme, conserved among low-GC, Gram-positive bacteria, that is involved in bacterial lipoprotein biosynthesis and synthesizes lyso-form lipoproteins. Its discovery is an essential first step in determining the physiological role of N-terminal lipoprotein acylation in Gram-positive bacteria and how these modifications impact bacterial cell envelope function.

Quantification of the d-Ala-d-Lac-Terminated Peptidoglycan Structure in Vancomycin-Resistant Enterococcus faecalis Using a Combined Solid-State Nuclear Magnetic Resonance and Mass Spectrometry Analysis.

Induction of vancomycin resistance in vancomycin-resistant enterococci (VRE) involves replacement of the d-Ala-d-Ala terminus of peptidoglycan (PG) stems with d-Ala-d-Lac, dramatically reducing the binding affinity of vancomycin for lipid II. Effects from vancomycin resistance induction in Enterococcus faecalis (ATCC 51299) were characterized using a combined solid-state nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. Solid-state NMR directly measured the total amounts of d-Lac and l,d-Ala metabolized from [2-13C]pyruvate, accumulated Park's nucleotide, and changes to the PG bridge-linking density during the early exponential growth phase (OD660 = 0.4) in intact whole cells of VRE. A high level of accumulation of depsipeptide-substituted Park's nucleotide consistent with the inhibition of the transglycosylation step of PG biosynthesis during the initial phase of vancomycin resistance was observed, while no changes to the PG bridge-linking density following the induction of vancomycin resistance were detected. This indicated that the attachment of the PG bridge to lipid II by the peptidyl transferases was not inhibited by the d-Ala-d-Lac-substituted PG stem structure in VRE. Compositions of mutanolysin-digested isolated cell walls of VRE grown with and without vancomycin resistance induction were determined by LC-MS. Muropeptides with PG stems terminating in d-Ala-d-Lac were found only in VRE grown in the presence of vancomycin. Percentages of muropeptides with a pentapeptide stem terminating in d-Ala-d-Lac for VRE grown in the presence of vancomycin were 26% for the midexponential phase (OD660 = 0.6) and 57% for the stationary growth phase (OD660 = 1.0). These high percentages indicate that d-Ala-d-Lac-substituted lipid II was efficiently utilized for PG biosynthesis in VRE.