scholarly journals A Fast, Efficient and Easy to Implement Method to Purify Bacterial Pili From Lacticaseibacillus rhamnosus GG Based on Multimodal Chromatography

2020 ◽  
Vol 11 ◽  
Author(s):  
Raphael Dos Santos Morais ◽  
Sofiane El-Kirat-Chatel ◽  
Jennifer Burgain ◽  
Blandine Simard ◽  
Sarah Barrau ◽  
...  
Keyword(s):  
Probiotic Strain ◽  
Uv Spectrum ◽  
Gram Positive ◽  
Laboratory Equipment ◽  
Gram Positive Bacteria ◽  
Multimodal Chromatography ◽  
Protein Partners ◽  
Sds Page ◽  
Simple Protocol ◽  
High Degree

Pili are polymeric proteins located at the cell surface of bacteria. These filamentous proteins play a pivotal role in bacterial adhesion with the surrounding environment. They are found both in Gram-negative and Gram-positive bacteria but differ in their structural organization. Purifying these high molecular weight proteins is challenging and has certainly slowed down their characterization. Here, we propose a chromatography-based protocol, mainly relying on multimodal chromatography (core bead technology using Capto Core 700 resin), to purify sortase-dependent SpaCBA pili from the probiotic strain Lacticaseibacillus rhamnosus GG (LGG). Contrary to previously published methods, this purification protocol does not require specific antibodies nor complex laboratory equipment, including for the multimodal chromatography step, and provides high degree of protein purity. No other proteins were detectable by SDS-PAGE and the 260/280 nm ratio (∼0.6) of the UV spectrum confirmed the absence of any other co-purified macromolecules. One can obtain ∼50 μg of purified pili, starting from 1 L culture at OD600nm ≈ 1, in 2–3 working days. This simple protocol could be useful to numerous laboratories to purify pili from LGG easily. Therefore, the present work should boost specific studies dedicated to LGG SpaCBA pili and the characterization of the interactions occurring with their protein partners at the molecular level. Moreover, this straightforward purification process might be extended to the purification of sortase-dependant pili from other Gram-positive bacteria.

scholarly journals Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments

2007 ◽  
Vol 73 (10) ◽  
pp. 3272-3282 ◽  
Author(s):  
Erin A. Gontang ◽  
William Fenical ◽  
Paul R. Jensen
Keyword(s):  
New Taxa ◽  
Marine Sediments ◽  
Sequence Data ◽  
Rrna Gene ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Operational Taxonomic Units ◽  
The Republic ◽  
Republic Of Palau ◽  
High Degree

ABSTRACT Major advances in our understanding of marine bacterial diversity have been gained through studies of bacterioplankton, the vast majority of which appear to be gram negative. Less effort has been devoted to studies of bacteria inhabiting marine sediments, yet there is evidence to suggest that gram-positive bacteria comprise a relatively large proportion of these communities. To further expand our understanding of the aerobic gram-positive bacteria present in tropical marine sediments, a culture-dependent approach was applied to sediments collected in the Republic of Palau from the intertidal zone to depths of 500 m. This investigation resulted in the isolation of 1,624 diverse gram-positive bacteria spanning 22 families, including many that appear to represent new taxa. Phylogenetic analysis of 189 representative isolates, based on 16S rRNA gene sequence data, indicated that 124 (65.6%) belonged to the class Actinobacteria while the remaining 65 (34.4%) were members of the class Bacilli. Using a sequence identity value of ≥98%, the 189 isolates grouped into 78 operational taxonomic units, of which 29 (37.2%) are likely to represent new taxa. The high degree of phylogenetic novelty observed during this study highlights the fact that a great deal remains to be learned about the diversity of gram-positive bacteria in marine sediments.

scholarly journals Functional Identification of Rubber Oxygenase (RoxA) in Soil and Marine Myxobacteria

2013 ◽  
Vol 79 (20) ◽  
pp. 6391-6399 ◽  
Author(s):  
Jakob Birke ◽  
Wolf Röther ◽  
Georg Schmitt ◽  
Dieter Jendrossek
Keyword(s):  
Chemical Reduction ◽  
Three Dimensional ◽  
Visible Spectrum ◽  
Functional Identification ◽  
Gram Positive ◽  
Zone Formation ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Degrading Bacteria ◽  
High Degree

ABSTRACTThe rubber oxygenase (RoxA) ofXanthomonassp. strain 35Y (RoxAXsp) is so far the only known extracellularc-type diheme cytochrome that is able to cleave poly(cis-1,4-isoprene). All other rubber-degrading bacteria described are Gram positive and employ a nonheme protein (latex-clearing protein [Lcp]) for the postulated primary attack of polyisoprene. Here, we identified RoxA orthologs in the genomes ofHaliangium ochraceum,Myxococcus fulvus,Corallococcus coralloides, andChondromyces apiculatus. TheroxAorthologs ofH. ochraceum(RoxAHoc),C. coralloidesBO35 (RoxACco), andM. fulvus(RoxAMfu) were functionally expressed in a ΔroxA Xanthomonassp. 35Y background. All RoxA orthologs oxidatively cleaved polyisoprene, as revealed by restoration of clearing-zone formation and detection of 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) as a cleavage product. RoxAXsp, RoxAMfu, and RoxACcowere purified and biochemically characterized. The optimal temperature of RoxACcoand RoxAMfuwas between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxACcoand RoxAMfuindicated the presence of two slightly different heme centers with absorption maxima between 549 and 553 nm, similar to RoxAXsp. Sequence analysis and modeling of the three-dimensional structures of the RoxA orthologs revealed a high degree of similarity to the recently solved RoxAXspstructure and included several conserved residues, notably, W302, F317, and a MauG motif at about H517. Lcp-like sequences were not detected in the genomes of theXanthomonassp. 35Y,H. ochraceum,M. fulvus, andC. coralloides. No RoxA orthologs were found in Gram-positive bacteria, and this first description of functional RoxA in Gram-negative bacteria other thanXanthomonasproves that RoxA is more common among rubber degraders than was previously assumed.

scholarly journals Two Spx Proteins Modulate Stress Tolerance, Survival, and Virulence in Streptococcus mutans

2010 ◽  
Vol 192 (10) ◽  
pp. 2546-2556 ◽  
Author(s):  
Jessica K. Kajfasz ◽  
Isamar Rivera-Ramos ◽  
Jacqueline Abranches ◽  
Alaina R. Martinez ◽  
Pedro L. Rosalen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Streptococcus Mutans ◽  
Gram Positive ◽  
Enzymatic Assays ◽  
Stress Genes ◽  
Gram Positive Bacteria ◽  
Underlying Mechanisms ◽  
Controlling Processes ◽  
High Degree

ABSTRACT Previous work suggested that the underlying mechanisms by which the Streptococcus mutans ClpXP protease affects virulence traits are associated with accumulation of two orthologues of the Spx regulator, named SpxA and SpxB. Here, a thorough characterization of strains lacking the spx genes (ΔspxA, ΔspxB, and ΔspxA ΔspxB) revealed that Spx, indeed, participates in the regulation of processes associated with S. mutans pathogenesis. The ΔspxA strain displayed impaired ability to grow under acidic and oxidative stress conditions and had diminished long-term viability at low pH. Although the ΔspxB strain did not show any inherent stress-sensitive phenotype, the phenotypes observed in ΔspxA were more pronounced in the ΔspxA ΔspxB double mutant. By using two in vivo models, we demonstrate for the first time that Spx is required for virulence in a Gram-positive pathogen. Microarrays confirmed the global regulatory role of SpxA and SpxB. In particular, SpxA was shown to positively regulate genes associated with oxidative stress, a finding supported by enzymatic assays. SpxB had a secondary role in regulation of oxidative stress genes but appeared to play a larger role in controlling processes associated with cell wall homeostasis. Given the high degree of conservation between Spx proteins of low-GC Gram-positive bacteria, these results are likely to have broad implications.

scholarly journals Tertiary Structure of Staphylococcus aureus Cell Wall Murein

2004 ◽  
Vol 186 (21) ◽  
pp. 7141-7148 ◽  
Author(s):  
Boris A. Dmitriev ◽  
Filip V. Toukach ◽  
O. Holst ◽  
E. T. Rietschel ◽  
S. Ehlers
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Tertiary Structure ◽  
Gram Positive ◽  
Division Plane ◽  
Gram Positive Bacteria ◽  
Computer Simulation Studies ◽  
High Degree ◽  
Division Plane Orientation

ABSTRACT The recently described scaffold model of murein architecture depicts the gram-negative bacterial cell wall as a gel-like matrix composed of cross-linked glycan strands oriented perpendicularly to the plasma membrane while peptide bridges adopt a parallel orientation (B. A. Dmitriev, F. V. Toukach, K. J. Schaper, O. Holst, E. T. Rietschel, and S. Ehlers, J. Bacteriol. 185:3458-3468, 2003). Based on the scaffold model, we now present computer simulation studies on the peptidoglycan arrangement of the gram-positive organism Staphylococcus aureus, which show that the orientation of peptide bridges is critical for the highly cross-linked murein architecture of this microorganism. According to the proposed refined model, staphylococcal murein is composed of glycan and oligopeptide chains, both running in a plane that is perpendicular to the plasma membrane, with oligopeptide chains adopting a zigzag conformation and zippering adjacent glycan strands along their lengths. In contrast to previous models of murein in gram-positive bacteria, this model reflects the high degree of cross-linking that is the hallmark of the staphylococcal cell wall and is compatible with distinguishing features of S. aureus cytokinesis such as the triple consecutive alteration of the division plane orientation and the strictly centripetal mode of septum closure.

scholarly journals Two Proteins From Snake Venom Have Potent Antibacterial Effects Against Bacillus Anthracis and Streptococcus Pneumoniae

2020 ◽  
Vol 14 (3) ◽  
pp. 139-144
Author(s):  
Mahboobeh Talebi Mehrdar ◽  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bacillus Anthracis ◽  
Snake Venom ◽  
Inhibitory Effects ◽  
Antibacterial Effects ◽  
Gram Positive ◽  
E Coli ◽  
Molecular Weights ◽  
Gram Positive Bacteria ◽  
Sds Page

Background: Antibacterial proteins are widely expressed in snake venoms. Previously, we have isolated two immunodominant proteins with molecular weights of 14 and 65 kD from the snake venom of Naja naja oxiana (N. oxiana). It was demonstrated that they had potent inhibitory effects against gram-positive bacteria, S. aureus and B. subtilis but were less effective against gram-negative bacteria, such as E. coli and P. aeruginosa. This study aimed at investigating the potential antibacterial effects of the two proteins against Bacillus anthracis and Streptococcus pneumoniae. Methods: The proteins were identified by SDS-PAGE and western blot analysis, and isolated by Gel Electrophoresis (Electro-elution). The antibacterial effects were tested against the strains of Bacillus anthracis and Streptococcus pneumoniae, using broth microdilution and disc-diffusion assays. For comparison, the antibacterial effects of standard antibiotics, such as Gentamicin, Ampicillin, Penicillin, Amoxicillin and Ciprofloxacin were also tested on the same B. anthracis and S. pneumoniae batches under identical laboratory conditions. Results: The two proteins showed high immunogenicity and strongly inhibited the growth of gram-positive bacteria, B. anthracis, and to a lesser extent S. pneumoniae. Conclusion: The isolated proteins demonstrated strong antibacterial effects against Gram-positive bacteria, B. anthracis and S. pneumoniae, in addition to their previously known effects against S. aureus and B. subtilis.

Influence of Cell Wall Composition on the Fossilisation of Bacteria and the Implications for the Search for Early Life Forms

1997 ◽  
Vol 161 ◽  
pp. 491-504 ◽  
Author(s):  
Frances Westall
Keyword(s):  
Cell Wall ◽  
Life Forms ◽  
Cation Binding ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Transmission Electron ◽  
Hydrothermal Sediments ◽  
Identification Of Bacteria ◽  
The Difference

AbstractThe oldest cell-like structures on Earth are preserved in silicified lagoonal, shallow sea or hydrothermal sediments, such as some Archean formations in Western Australia and South Africa. Previous studies concentrated on the search for organic fossils in Archean rocks. Observations of silicified bacteria (as silica minerals) are scarce for both the Precambrian and the Phanerozoic, but reports of mineral bacteria finds, in general, are increasing. The problems associated with the identification of authentic fossil bacteria and, if possible, closer identification of bacteria type can, in part, be overcome by experimental fossilisation studies. These have shown that not all bacteria fossilise in the same way and, indeed, some seem to be very resistent to fossilisation. This paper deals with a transmission electron microscope investigation of the silicification of four species of bacteria commonly found in the environment. The Gram positiveBacillus laterosporusand its spore produced a robust, durable crust upon silicification, whereas the Gram negativePseudomonas fluorescens, Ps. vesicularis, andPs. acidovoranspresented delicately preserved walls. The greater amount of peptidoglycan, containing abundant metal cation binding sites, in the cell wall of the Gram positive bacterium, probably accounts for the difference in the mode of fossilisation. The Gram positive bacteria are, therefore, probably most likely to be preserved in the terrestrial and extraterrestrial rock record.

Microanatomy of the Periplasm of Bacillus Licheniformis

1971 ◽  
Vol 29 ◽  
pp. 262-263
Author(s):  
B.K. Ghosh
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Bacillus Licheniformis ◽  
External Environment ◽  
Permeability Barrier ◽  
Periplasmic Space ◽  
Modified Technique ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

Rapid demonstration of septic or infectious arthritis due to Gram-negative bacteria

1992 ◽  
Vol 50 (1) ◽  
pp. 686-687
Author(s):  
Jacob S. Hanker ◽  
Paul R. Gross ◽  
Beverly L. Giammara
Keyword(s):  
Chronic Arthritis ◽  
Blood Cultures ◽  
Gram Negative Bacteria ◽  
Infectious Arthritis ◽  
Bacterial Arthritis ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Blood cultures are positive in approximately only 50 per cent of the patients with nongonococcal bacterial infectious arthritis and about 20 per cent of those with gonococcal arthritis. But the concept that gram-negative bacteria could be involved even in chronic arthritis is well-supported. Gram stains are more definitive in staphylococcal arthritis caused by gram-positive bacteria than in bacterial arthritis due to gram-negative bacteria. In the latter situation where gram-negative bacilli are the problem, Gram stains are helpful for 50% of the patients; they are only helpful for 25% of the patients, however, where gram-negative gonococci are the problem. In arthritis due to gram-positive Staphylococci. Gramstained smears are positive for 75% of the patients.

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