scholarly journals The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan

2020 ◽  
Vol 295 (15) ◽  
pp. 5110-5123 ◽  
Author(s):  
Lin Shen ◽  
Albertus Viljoen ◽  
Sydney Villaume ◽  
Maju Joe ◽  
Iman Halloum ◽  
...  
Keyword(s):  
Cell Wall ◽  
Structural Changes ◽  
Cell Envelope ◽  
Wall Structure ◽  
Tubercle Bacillus ◽  
Bioinformatics Analyses ◽  
The Past ◽  
Biochemical Assays ◽  
Mycobacterial Protein

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

Lignin Reactions and Structural Alternations under Typical Biomass Pretreatment Methods

2019 ◽  
Vol 23 (20) ◽  
pp. 2145-2154 ◽  
Author(s):  
Linjiang Zhu ◽  
Anjie Xu ◽  
Hui Zhang ◽  
Yuele Lu ◽  
Shijie Liu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Chemical Structure ◽  
Structural Changes ◽  
Physical Structure ◽  
Refining Process ◽  
Biomass Pretreatment ◽  
Wall Structure ◽  
The Past ◽  
Agriculture Residues ◽  
Biological Methods

The utilization of biomass in the production of renewable bioenergy and biomaterials has been a popular topic since the past decades as they are rich in carbohydrates. Most biomasses, such as wood, monocotyledons, and agriculture residues, need to be pretreated before the conversion of carbohydrates in order to break down the recalcitrant cell wall structure and increase the fiber accessibility. To date, a variety of pretreatment methods have been developed that vary from physical to chemical and biological methods. Pretreatment processes affect the cell wall physical structure as well as the chemical structure of the cell wall constituents. Comparing to the studies of the cellulose and hemicelluloses structural changes during pretreatment, such studies on lignin are relatively limited. On the other hand, in order to utilize the part of lignin from biorefinery processes, the understanding of the lignin structural changes during the refining process becomes important. In this study, typical pretreatment methods such as hydrothermal pretreatment, alkaline pretreatment, biodegradation, and oxidative pretreatment are introduced and their corresponding impacts on the lignin structures are reviewed.

scholarly journals Characterization of Monolaurin Resistance in Enterococcus faecalis

2007 ◽  
Vol 73 (17) ◽  
pp. 5507-5515 ◽  
Author(s):  
Muriel Dufour ◽  
Janet M. Manson ◽  
Philip J. Bremer ◽  
Jean-Pierre Dufour ◽  
Gregory M. Cook ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Wall Structure ◽  
Transmission Electron ◽  
Serious Infections ◽  
Autolytic Activity

ABSTRACT There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 μg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

scholarly journals Combined whole cell wall analysis and streamlined in silico carbohydrate-active enzyme discovery to improve biocatalytic conversion of agricultural crop residues

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jeffrey P. Tingley ◽  
Kristin E. Low ◽  
Xiaohui Xing ◽  
D. Wade Abbott
Keyword(s):  
Cell Wall ◽  
Microbial Communities ◽  
Cell Walls ◽  
Crop Residues ◽  
Monosaccharide Composition ◽  
Wall Structure ◽  
Agricultural Crop ◽  
Fine Chemistry ◽  
Fermentable Carbohydrates

AbstractThe production of biofuels as an efficient source of renewable energy has received considerable attention due to increasing energy demands and regulatory incentives to reduce greenhouse gas emissions. Second-generation biofuel feedstocks, including agricultural crop residues generated on-farm during annual harvests, are abundant, inexpensive, and sustainable. Unlike first-generation feedstocks, which are enriched in easily fermentable carbohydrates, crop residue cell walls are highly resistant to saccharification, fermentation, and valorization. Crop residues contain recalcitrant polysaccharides, including cellulose, hemicelluloses, pectins, and lignin and lignin-carbohydrate complexes. In addition, their cell walls can vary in linkage structure and monosaccharide composition between plant sources. Characterization of total cell wall structure, including high-resolution analyses of saccharide composition, linkage, and complex structures using chromatography-based methods, nuclear magnetic resonance, -omics, and antibody glycome profiling, provides critical insight into the fine chemistry of feedstock cell walls. Furthermore, improving both the catalytic potential of microbial communities that populate biodigester reactors and the efficiency of pre-treatments used in bioethanol production may improve bioconversion rates and yields. Toward this end, knowledge and characterization of carbohydrate-active enzymes (CAZymes) involved in dynamic biomass deconstruction is pivotal. Here we overview the use of common “-omics”-based methods for the study of lignocellulose-metabolizing communities and microorganisms, as well as methods for annotation and discovery of CAZymes, and accurate prediction of CAZyme function. Emerging approaches for analysis of large datasets, including metagenome-assembled genomes, are also discussed. Using complementary glycomic and meta-omic methods to characterize agricultural residues and the microbial communities that digest them provides promising streams of research to maximize value and energy extraction from crop waste streams.

scholarly journals Genomic Characterization of Candidate Division LCP-89 Reveals an Atypical Cell Wall Structure, Microcompartment Production, and Dual Respiratory and Fermentative Capacities

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Noha H. Youssef ◽  
Ibrahim F. Farag ◽  
C. Ryan Hahn ◽  
Jessica Jarett ◽  
Eric Becraft ◽  
...  
Keyword(s):  
Cell Wall ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Structural Features ◽  
Wall Structure ◽  
Rrna Gene ◽  
Atypical Cell ◽  
Wide Range ◽  
Candidate Division

ABSTRACTRecent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla (Calditrichota, SM32-31, AABM5-125-24, and KSB1) within the broader FCB (Fibrobacteres-Chlorobi-Bacteroidetes) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genome-based phylogenies.IMPORTANCEOur understanding of the metabolic capacities, physiological preferences, and ecological roles of yet-uncultured microbial phyla is expanding rapidly. Two distinct approaches are currently being utilized for characterizing microbial communities in nature: amplicon-based 16S rRNA gene surveys for community characterization and metagenomics/single-cell genomics for detailed metabolic reconstruction. The occurrence of multiple yet-uncultured bacterial phyla has been documented using 16S rRNA surveys, and obtaining genome representatives of these yet-uncultured lineages is critical to our understanding of the role of yet-uncultured organisms in nature. This study provides a genomics-based analysis highlighting the structural features and metabolic capacities of a yet-uncultured bacterial phylum (LCP-89) previously identified in 16S rRNA surveys for which no prior genomes have been described. Our analysis identifies several interesting structural features for members of this phylum, e.g., lack of peptidoglycan biosynthetic machinery and the ability to form bacterial microcompartments. Predicted metabolic capabilities include degradation of a wide range of sugars, anaerobic respiratory capacity, and fermentative capacities. In addition to the detailed structural and metabolic analysis provided for candidate division LCP-89, this effort represents an additional step toward a unified scheme for microbial taxonomy by reconciling 16S rRNA gene-based and genomics-based taxonomic outlines.

Li+ effect on the cell wall of the yeast Saccharomyces cerevisiae as probed by FT-IR spectroscopy

2013 ◽  
Vol 8 (8) ◽  
pp. 724-729 ◽  
Author(s):  
Aurelijus Zimkus ◽  
Audrius Misiūnas ◽  
Larisa Chaustova
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Ir Spectroscopy ◽  
Absorption Band ◽  
Structural Changes ◽  
Yeast Cells ◽  
Wall Structure ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ft Ir ◽  
Ft Ir Spectroscopy

AbstractThe effect of Li+ ions as a transformation inducing agent on the yeast cell wall has been studied. Two Saccharomyces cerevisiae strains, p63-DC5 with a native cell wall, and strain XCY42-30D(mnn1) which contains structural changes in the mannan-protein complex, were used. Fourier transform infrared (FT-IR) spectroscopy has been used for the characterization of the yeast strains and for determination of the effect of lithium cations on the cell wall. A comparison of the carbohydrate absorption band positions in the 970–1185 cm−1 range, of Na+ and Li+ treated yeast cells has been estimated. Absorption band positions of the cell wall carbohydrates of p63-DC5 were not influenced by the studied ions. On the contrary, the treatment of XCY42-30D(mnn1) cells with Li+ ions shifted glucan band positions, implying that the cell wall structure of strain XCY42-30D(mnn1) is more sensitive to Li+ ion treatment.

scholarly journals Genome-wide identification of novel genes involved in Corynebacteriales cell envelope biogenesis using Corynebacterium glutamicum as a model

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0240497
Author(s):  
Célia de Sousa-d’Auria ◽  
Florence Constantinesco-Becker ◽  
Patricia Constant ◽  
Maryelle Tropis ◽  
Christine Houssin
Keyword(s):  
Cell Wall ◽  
Corynebacterium Glutamicum ◽  
Acid Metabolism ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Bioinformatics Analyses ◽  
Mycolic Acids ◽  
Genome Wide ◽  
Altered Cell ◽  
Covalent Complex

Corynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-cell wall antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.

scholarly journals Characterization of fmtA, a Gene That Modulates the Expression of Methicillin Resistance in Staphylococcus aureus

1999 ◽  
Vol 43 (9) ◽  
pp. 2121-2125 ◽  
Author(s):  
Hitoshi Komatsuzawa ◽  
Kouji Ohta ◽  
Harald Labischinski ◽  
Motoyuki Sugai ◽  
Hidekazu Suginaka
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Methicillin Resistance ◽  
Binding Activity ◽  
Wall Structure ◽  
Penicillin Binding Protein ◽  
Resistance Level ◽  
Penicillin Binding Proteins ◽  
Chromatography Analysis

ABSTRACT FmtA is a factor which affects the methicillin resistance level in methicillin-resistant Staphylococcus aureus. Since FmtA has two of three conserved motifs which are typically found in penicillin-binding proteins (PBPs) and β-lactamases, we investigated the penicillin-binding activity of recombinant FmtA and found no such activity. Immunoblotting analysis revealed that FmtA localizes in the membrane fraction. To investigate the function of FmtA, high-pressure liquid chromatography analysis of cell wall muropeptides was performed with an fmtA-inactivated mutant and its parent. The mutant showed a reduced cross-linking and partially reduced amidation of glutamate residues in the peptidoglycan of the mutant. The transcription of fmtA was dose dependently increased by the addition of β-lactam antibiotics, fosfomycin, and bacitracin, while its transcription was not changed by the addition of vancomycin or tetracycline. These results reveal that Fmt is a membrane-located, non-penicillin-binding protein and that mutation of fmtAaffects the cell wall structure, although its precise function is still unknown.

scholarly journals Transcription Factor Efg1 Shows a Haploinsufficiency Phenotype in Modulating the Cell Wall Architecture and Immunogenicity of Candida albicans

2011 ◽  
Vol 11 (2) ◽  
pp. 129-140 ◽  
Author(s):  
Martin Zavrel ◽  
Olivia Majer ◽  
Karl Kuchler ◽  
Steffen Rupp
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Structural Changes ◽  
Protein Composition ◽  
Surface Proteins ◽  
Wall Structure ◽  
Content Type ◽  
Cell Wall Biogenesis ◽  
General Metabolism

ABSTRACTTheCandida albicanstranscription factor Efg1 is known to be involved in many different cellular processes, including morphogenesis, general metabolism, and virulence. Here we show that besides its manifold roles, Efg1 also has a prominent effect on cell wall structure and composition, strongly affecting the structural glucan part. Deletion of only one allele ofEFG1already results in severe phenotypes for cell wall biogenesis, comparable to those with deletion of both alleles, indicative of a severe haploinsufficiency forEFG1. The observed defects in structural setup of the cell wall, together with previously reported alterations in expression of cell surface proteins, result in altered immunogenic properties of strains with compromised Efg1 function. This is shown by interaction studies with macrophages and primary dendritic cells. The structural changes in the cell wall carbohydrate meshwork presented here, together with the manifold changes in cell wall protein composition and metabolism reported in other studies, contribute to the altered immune response mounted by innate immune cells and to the altered virulence phenotypes observed for strains lackingEFG1.

scholarly journals Genome-wide identification of novel genes involved in Corynebacteriales cell envelope biogenesis using Corynebacterium glutamicum as a model

2020 ◽  
Author(s):  
Célia de Sousa-d’Auria ◽  
Florence Constantinesco-Becker ◽  
Patricia Constant ◽  
Maryelle Tropis ◽  
Christine Houssin
Keyword(s):  
Cell Wall ◽  
Corynebacterium Glutamicum ◽  
Acid Metabolism ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Bioinformatics Analyses ◽  
Mycolic Acids ◽  
Genome Wide ◽  
Altered Cell ◽  
Covalent Complex

AbstractCorynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-arabinogalactan antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.

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