High-throughput,Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.     

Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli

Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.     

Cell wall structure and function in lactic acid bacteria

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.     

Cell division and peptidoglycan assembly in Eschenchia coli

Research on bacterial cell division has recently gained renewed impetus because of new information about peptidoglycan assembly and about specific cell-division genes and their products. This paper concerns aspects of cell division that specifically concern the peptidoglycan. It is shown that upon division, peptidoglycan assembly switches from lateral wall location to the cell centre, that assembly takes place at the leading edge of the invaginating constriction, that the mode of glycan strand insertion changes from a single-stranded mode to a multi-stranded mode, and that the initiation of division (in contrast to its continuation) requires penicillin-insensitive peptidoglycan synthesis (PIPS). A membrane component X (possibly FtsQ) is proposed to coordinate PIPS with the cell division-initiating protein FtsZ. It is suggested that a largely proteinaceous macromolecular complex (divisome) at the leading edge of constriction encompasses three compartments (cytoplasm, membrane and periplasm). The composition of this complex is proposed to vary depending on whether division is being initiated or completed.     

Taking shape: control of bacterial cell wall biosynthesis

The characteristic shape of a bacterial cell is a function of the three dimensional architectures of the cell envelope and is determined by the balance between lateral wall extension and synthesis of peptidoglycan at the division septum. The three dimensional patterns of cell wall synthesis in the bacterium Bacillus subtilis is influenced by actin-like proteins that form helical coils in the cell and by the MreCD membrane proteins that link the cytoskeletal elements with the penicillin-binding proteins that carry out peptidoglycan synthesis. Recent genetic studies have provided important clues as to how these proteins are arranged in the cell and how they function to regulate cell shape.     

青海循化线辣椒中氨基酸含量分析

采用柱前衍生高效液相色谱法对循化线辣椒中精氨酸、丙氨酸、丝氨酸等17种氨基酸的含量进行测定。结果表明:线辣椒所含17种氨基酸中,必需氨基酸含量合理,利于人体吸收。     

大苞藤黄化学成分研究及其互变异构体超高效液相色谱质谱联用分析

从大苞藤黄枝叶的混合粉碎物中分离到11个化合物,运用光谱手段分别鉴定为neobractatin(1),brasixanthone B (2),5-O-methylxanthone V1 (3),10-O-methylmacluraxanthone (4),isobractatin (5),xanthone V1(6),xerophenone A (7),xerophenone B (8),bractatin (9),macluraxanthone (10)和3-O-methylneobractatin (11).本文首次应用超高效液相色谱-质谱联用技术分离了异构体7和8并测定了其精确分子量.其中化合物6～8为首次从该植物中发现.     

Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane

Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.     

反相高效液相色谱法测定厌氧菌有机酸代谢产物

建立反相高效液相色谱测定厌氧菌代谢发酵有机酸产物(乙酸、乳酸)的方法并用于测定乳酸菌代谢发酵产物中的含量。反相高效液相方法是一种简单、准确、灵敏的方法,可用于同时定量测定厌氧菌的有机酸代谢产物。     

CELL WALL AND PEPTIDOGLYCAN FROM Lactobacillus fermenti

Cell walls from Lactobacillus fermenti were prepared by differential centrifugation of disrupted cells, with and without trypsin treatment. Approximately 16% of the dry weight of walls was found in a crude trichloroacetic acid extract of the walls; half of this amount remained upon further purification. The purufied extract lacked alanine, but contained substantial amounts of glucosamine. The walls constituted 23 to 33% of the dry weight of the cell. The chemical composition of the various types of wall preparations and of the peptidoglycan from them was studied. The peptidoglycan contained equimolar proportions of glucosamine, muramic acid, l-alanine, d-glutamic acid, and lysine, with somewhat lower proportions of d-aspartic acid and d-alanine. The chemical composition of the peptidoglycan is similar to that reported for three other lactobacilli. In addition to the major constituents of walls and peptidoglycan, there were several minor amino acids. The protein and the amounts of the minor amino acids decreased, and among these threonine and arginine were completely absent from preparations obtained with trypsin. Such preparations contained higher proportions of the d-isomers of alanine, glutamic acid, and aspartic acid as compared to walls and peptidoglycan prepared without trypsin. In addition, walls isolated with the use of trypsin were susceptible to lysozyme, whereas those prepared without trypsin were not. However, the trypsin treatment did not result in any change of the ultrastructure as revealed by electron microscope studies.     

Subunit Cell Wall of Sulfolobus acidocaldarius

The cell wall of Sulfolobus acidocaldarius has been isolated. Cells were mechanically disrupted with a French press, and the cytoplasmic membrane was removed by extracting cell-envelope fragments with Triton X-100. The Triton-insoluble cell wall material retained the characteristic subunit structure when examined in the electron microscope. Isolated cell wall fragments formed in open sheets that were easily separated from cytoplasmic contamination. Chemical studies showed that the Triton-insoluble cell wall fragments consisted of lipoprotein with small amounts of carbohydrate and hexosamine. The amino acid composition indicated a highly charged hydrophobic cell surface. The presence of diaminopimelic acid with only traces of muramic acid indicates that the cell envelope does not have a rigid peptidoglycan layer. The results of chemical analyses and electron microscopy suggest a wall-membrane interaction stabilizing the cell envelope. The chemical and physical properties of this type of cell envelope would appear to form the basis for a new major division of bacteria with the definitive characteristics of a morphologically distinct subunit cell wall devoid of peptidoglycan.     

Breaking strengths of pollen grain walls

50% breaking point pressures of pollen grain walls of eleven species were determined. The breaking point pressures of most pollen grain walls are equivalent to those reported in the literature for other types of living cell walls such as bacterial spore coats, algal cell walls, mold sporophyte cells, and dicot suspension culture cells. The strongest pollen grain walls are two or three orders of magnitude stronger, however. Pollen grain walls are proportionately very thick in comparison to other types of cell walls. It is this thickness, not the construction or physical properties of the pollen grain wall, that most probably accounts for their strength.     

Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems

Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis, PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.     

Genomic channeling in bacterial cell division

The bacterial dcw cluster is a group of genes involved in cell division and peptidoglycan synthesis. Comparison of the cluster across several bacterial genomes shows that its gene content and its gene order are conserved in distant bacterial lineages and, moreover, that, being most conserved in rod-shaped bacteria, the degree of conservation relates to bacterial morphology. We propose a model in which the selective pressure to maintain the cluster arises from the need to efficiently coordinate the processes of elongation and septation in rod-shaped bacteria. Gene order in the dcw cluster would be conserved as a result of mechanisms comprising: (i) a limited amount of peptidoglycan precursors required both for septation and elongation of the wall; (ii) co-translational assembly of the protein complexes involved in cell division and in the synthesis of the peptidoglycan precursors; and (iii) alternation in the cellular localization of the assembled complexes to participate either in the synthesis of the septal peptidoglycan and division, or in the synthesis of the lateral wall. The name genomic channeling is proposed for this model as it involves a genomic arrangement that could facilitate the assembly of specific protein complexes and their subsequent conveyance to specific locations in the crowded cytoplasm and the envelope.     

Species dependency of in vitro macrophage activation by bacterial peptidoglycans.

The effect of various bacterial cell wall components on in vitro biological function of murine peritoneal exudate macrophages was evaluated. We examined four different parameters of metabolic activity and monokine secretion. Peritoneal exudate macrophages from rats and guinea pigs, all of the strains tested, were stimulated by whole bacterial cell wall preparations, purified bacterial cell wall peptidoglucans, its water-soluble peptidolglycan fragments, muramyl dipeptides and amphipathic substances. Murine peritoneal exudate macrophages were activated by amphipathic substances of gram-positive bacteria. However, macrophages from mice, irrespective of strains, were not stimulated in the in vitro assay systems by purified bacterial cell wall peptidoglycan, water-soluble bacterial peptidoglycan fragments or muramyl dipeptides. These results suggest that macrophage activation by bacterial peptidoglycan in vitro is animal species specific.     

HPLC Retention time prediction for metabolome analysi

Liquid Chromatography Time-of-Flight Mass Spectrometry (LC-TOF-MS) is widely used for profiling metabolite compounds. LC-TOF-MS is a chemical analysis technique that combines the physical separation capabilities of high-pressure liquid chromatography (HPLC) with the mass analysis capabilities of Time-of-Flight Mass Spectrometry (TOF-MS) which utilizes the difference in the flight time of ions due to difference in the mass-to-charge ratio. Since metabolite compounds have various chemical characteristics, their precise identification is a crucial problem of metabolomics research. Contemporaneously analyzed reference standards are commonly required for mass spectral matching and retention time matching, but there are far fewer reference standards than there are compounds in the organism. We therefore developed a retention time prediction method for HPLC to improve the accuracy of identification of metabolite compounds. This method uses a combination of Support Vector Regression and Multiple Linear Regression adaptively to the measured retention time. We achieved a strong correlation (correlation coefficient = 0.974) between measured and predicted retention times for our experimental data. We also demonstrated a successful identification of an E. coli metabolite compound that cannot be identified by precise mass alone.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

Halococcus morrhuae: A sulfated heteropolysaccharide as the structural component of the bacterial cell wall

The qualitative and quantitative composition of purified cell walls of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heteroglycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.     

Monitoring surface chemical changes in the bacterial cell wall: multivariate analysis of cryo-x-ray photoelectron spectroscopy data

Gram-negative bacteria can alter the composition of the lipopolysaccharide (LPS) layer of the outer membrane as a response to different growth conditions and external stimuli. These alterations can, for example, promote attachment to surfaces and biofilm formation. The changes occur in the outermost layer of the cell and may consequently influence interactions between bacterial cells and surrounding host tissue, as well as other surfaces. Microscopic analyses, fractionation of bacterial cells, or other traditional microbiological assays have previously been used to study these alterations. These methods can, however, be time consuming and do not always give detailed chemical information about the bacterial cell surface. We here present an analytical method that provides chemical information on the outermost portion of bacterial cells with respect to protein, peptidoglycan, lipid, and polysaccharide content. The method involves cryo-x-ray photoelectron spectroscopy analyses of the outermost portion (within ～10 nm of the surface) of intact bacterial cells followed by a multivariate curve resolution analysis of carbon spectra. It can be used as a tool for characterizing and monitoring variations in the chemical composition of bacterial cell walls or of isolated outer membrane vesicles, variations that result from e.g. mutations or external stimuli. The method enabled us to predict accurately the alterations in polysaccharide content and surface chemistries of a set of well characterized Escherichia coli LPS mutants. The described approach may moreover be applied to monitor surface chemical composition of other biological samples.     

乳酸菌肽聚糖的研究进展

肽聚糖是乳酸菌细胞壁的必需成分,它的化学结构较为保守固定,而其合成是一个涉及多步反应的复杂过程。乳酸菌肽聚糖具有多种生物学活性,比如免疫增强功能、抗感染、抗肿瘤及抗过敏等。本文对乳酸菌肽聚糖的组成结构和生物学活性进行了简要的介绍,重点综述了近年来乳酸菌肽聚糖代谢及其调控过程的研究进展,并指出了乳酸菌肽聚糖未来研究的方向。