Identification of Gut Bacteria such as Lactobacillus johnsonii that Disseminate to Systemic Tissues of Wild Type and MyD88–/– Mice

ABSTRACT Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides—a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsonii. IMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

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Conspecific coprophagy stimulates normal development in a germ-free model invertebrate

PeerJ ◽

10.7717/peerj.6914 ◽

2019 ◽

Vol 7 ◽

pp. e6914 ◽

Cited By ~ 6

Author(s):

Benjamin C. Jahnes ◽

Madeline Herrmann ◽

Zakee L. Sabree

Keyword(s):

Periplaneta Americana ◽

Model System ◽

Gut Bacteria ◽

American Cockroach ◽

Wild Type ◽

Germ Free ◽

Primary Means ◽

Invertebrate Model ◽

Nutrient Provisioning ◽

Organismal Development

Microbial assemblages residing within and on animal gastric tissues contribute to various host beneficial processes that include diet accessibility and nutrient provisioning, and we sought to examine the degree to which intergenerational and community-acquired gut bacteria impact development in a tractable germ-free (GF) invertebrate model system. Coprophagy is a common behavior in cockroaches and termites that provides access to both nutrients and the primary means by which juveniles are inoculated with beneficial gut bacteria. This hypothesis was tested in the American cockroach (Periplaneta americana) by interfering with this means of acquiring gut bacteria, which resulted in GF insects that exhibited prolonged growth rates and gut tissue dysmorphias relative to wild-type (WT)P. americana. Conventionalization of GFP. americanavia consumption of frass (feces) from conspecifics and siblings reared under non-sterile conditions resulted in colonization ofP. americanagut tissues by a diverse microbial community and a significant (p< 0.05) recovery of WT level growth and hindgut tissue development phenotypes. These data suggest that coprophagy is essential for normal gut tissue and organismal development by introducing beneficial gut bacteria toP. americana, and that the GFP. americanamodel system is a useful system for examining how gut bacteria impact host outcomes.

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Enriched environmental conditions modify the gut microbiome composition and fecal markers of inflammation in Parkinson’s disease

10.1101/614834 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yogesh Singh ◽

Mohamed El Hadidi ◽

Jakob Matthes ◽

Zinah Wassouf ◽

Julia M. Schulze-Hentrich ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Transgenic Mice ◽

Environmental Conditions ◽

Gut Microbiome ◽

Gut Bacteria ◽

Dietary Factors ◽

Enriched Environment ◽

Wild Type ◽

Markers Of Inflammation

AbstractBackgroundRecent findings suggest an implication of the gut microbiome in Parkinson’s disease patients. Parkinson’s disease onset and progression has also been linked with various environmental factors such as physical activity, exposure to pesticides, head injury, nicotine, and dietary factors.ObjectivesIn this study, we used a transgenic mouse model overexpressing the complete human SNCA genes modeling familial and sporadic forms of Parkinson’s disease to study whether environmental conditions such as standard versus enriched environment changes the gut microbiome and influences disease progression.MethodsWe performed 16S rRNA DNA sequencing on fecal samples for microbiome analysis and studied fecal inflammatory calprotectin from the colon of control and transgenic mice kept under standard environment and enriched environment conditions.ResultsThe overall composition of the gut microbiota was not changed in transgenic mice compared with wild-type in enriched environment, however, individual gut bacteria at genus level such as Lactobacillus sp. were significantly changed in transgenic mice. Furthermore, enriched environment significantly reduced colon fecal inflammatory calprotectin protein in wild-type and transgenic enriched environment conditions compared to standard environment.ConclusionOur data suggest that enriched social environment has a positive effect on the induction of SNCA mediated inflammation in the intestine by changing anti-inflammatory gut bacteria.

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Neurotropic enteroviruses co-opt “fair-weather-friend” commensal gut microbiota to drive host infection and central nervous system disturbances

Behavioral and Brain Sciences ◽

10.1017/s0140525x18002741 ◽

2019 ◽

Vol 42 ◽

Author(s):

Kevin B. Clark

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Gut Bacteria ◽

Infection Cycle ◽

Fair Weather ◽

Host Health ◽

Microbe Interactions ◽

Animal Hosts ◽

Host Infection ◽

Neuropsychiatric Conditions

Abstract Some neurotropic enteroviruses hijack Trojan horse/raft commensal gut bacteria to render devastating biomimicking cryptic attacks on human/animal hosts. Such virus-microbe interactions manipulate hosts’ gut-brain axes with accompanying infection-cycle-optimizing central nervous system (CNS) disturbances, including severe neurodevelopmental, neuromotor, and neuropsychiatric conditions. Co-opted bacteria thus indirectly influence host health, development, behavior, and mind as possible “fair-weather-friend” symbionts, switching from commensal to context-dependent pathogen-like strategies benefiting gut-bacteria fitness.

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Ultrastructure of Melanin Formation in Curvularia sp., Alternaria sp., and Drechslera Sorokiniana

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079449 ◽

1977 ◽

Vol 35 ◽

pp. 398-399

Author(s):

M. H. Wheeler ◽

W. J. Tolmsoff ◽

A. A. Bell

Keyword(s):

Potassium Phosphate ◽

Thielaviopsis Basicola ◽

Wild Type ◽

Potassium Phosphate Buffer ◽

Transmission Microscopy ◽

Electron Transmission ◽

Melanin Formation ◽

Before And After ◽

Alternaria Sp ◽

Electron Transmission Microscopy

(+)-Scytalone [3,4-dihydro-3,6,8-trihydroxy-l-(2Hj-naphthalenone] and 1,8-di- hydroxynaphthalene (DHN) have been proposed as intermediates of melanin synthesis in the fungi Verticillium dahliae (1, 2, 3, 4) and Thielaviopsis basicola (4, 5). Scytalone is enzymatically dehydrated by V. dahliae to 1,3,8-trihydroxynaphthalene which is then reduced to (-)-vermelone [(-)-3,4- dihydro-3,8-dihydroxy-1(2H)-naphthalenone]. Vermelone is subsequently dehydrated to DHN which is enzymatically polymerized to melanin.Melanin formation in Curvularia sp., Alternaria sp., and Drechslera soro- kiniana was examined by light and electron-transmission microscopy. Wild-type isolates of each fungus were compared with albino mutants before and after treatment with 1 mM scytalone or 0.1 mM DHN in 50 mM potassium phosphate buffer, pH 7.0. Both chemicals were converted to dark pigments in the walls of hyphae and conidia of the albino mutants. The darkened cells were similar in appearance to corresponding cells of the wild types under the light microscope.

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Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090257 ◽

1976 ◽

Vol 34 ◽

pp. 54-55

Author(s):

Karen S. Howard ◽

H. D. Braymer ◽

M. D. Socolofsky ◽

S. A. Milligan

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Wild Type ◽

Morphological Comparison ◽

Small Areas ◽

Solid Media ◽

Thin Sectioning ◽

X Cells ◽

Mutant Cells ◽

Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

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Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160996 ◽

1990 ◽

Vol 48 (3) ◽

pp. 688-689

Author(s):

Thecan Caesar-Ton That ◽

Lynn Epstein

Keyword(s):

Freeze Substitution ◽

Uranyl Acetate ◽

Wild Type ◽

Nectria Haematococca ◽

Fruit Extract ◽

Dialysis Tubing ◽

Tube Emergence ◽

Contrast Microscopy ◽

In The Wild ◽

Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

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Identification of a baculovirus polyhedron formation mutant with a novel phenotype

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166440 ◽

1996 ◽

Vol 54 ◽

pp. 796-797

Author(s):

James M. Slavicek ◽

Melissa J. Mercer ◽

Mary Ellen Kelly

Keyword(s):

Viral Gene ◽

Gene Products ◽

Type Number ◽

Infection Cycle ◽

Wild Type ◽

Protein Matrix ◽

Insect Midgut ◽

Viral Particles ◽

Budded Virus ◽

Viral Form

Nucleopolyhedroviruses (NPV, family Baculoviridae) produce two morphological forms, a budded virus form and a viral form that is occluded into a paracrystalline protein matrix. This structure is termed a polyhedron and is composed primarily of the protein polyhedrin. Insects are infected by NPVs after ingestion of the polyhedron and release of the occluded virions through dissolution of the polyhedron in the alkaline environment of the insect midgut. Early after infection the budded virus form is produced. It buds through the plasma membrane and then infects other cells. Later in the infection cycle the occluded form of the virus is generated (reviewed by Blissard and Rohrmann, 1990).The processes of polyhedron formation and virion occlusion are likely to involve a number of viral gene products. However, only two genes, the polyhedrin gene and 25K FP gene, have been identified to date that are necessary for the wild type number of polyhedra to be formed and viral particles occluded.

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Arabidopsis 4-COUMAROYL-COA LIGASE 8 contributes to the biosynthesis of the benzenoid ring of coenzyme Q in peroxisomes

Biochemical Journal ◽

10.1042/bcj20190688 ◽

2019 ◽

Vol 476 (22) ◽

pp. 3521-3532

Author(s):

Eric Soubeyrand ◽

Megan Kelly ◽

Shea A. Keene ◽

Ann C. Bernert ◽

Scott Latimer ◽

...

Keyword(s):

Oxidative Metabolism ◽

Time Course ◽

Reverse Genetics ◽

De Novo ◽

Coenzyme Q ◽

Control Level ◽

Wild Type ◽

Fluorescent Reporters ◽

Coa Ligase ◽

Peroxisomal Targeting

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

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