Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization

1998 ◽  
Vol 64 (8) ◽  
pp. 3059-3062 ◽  
Author(s):  
Elena Bidnenko ◽  
Carine Mercier ◽  
Josselyne Tremblay ◽  
Patrick Tailliez ◽  
Saulius Kulakauskas
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Cell Walls ◽  
Fluorescent In Situ Hybridization ◽  
Cell Level ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Identification Of Bacteria

ABSTRACT Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.

Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH)

2009 ◽  
Vol 55 (4) ◽  
pp. 465-472 ◽  
Author(s):  
Ryohei Ueno
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Cell Walls ◽  
Fluorescent In Situ Hybridization ◽  
Rapid Identification ◽  
Oligonucleotide Probes ◽  
Logarithmic Growth Phase ◽  
Prototheca Wickerhamii ◽  
Algal Genus

Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

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.

scholarly journals L-Ficolin Specifically Binds to Lipoteichoic Acid, a Cell Wall Constituent of Gram-Positive Bacteria, and Activates the Lectin Pathway of Complement

2004 ◽  
Vol 172 (2) ◽  
pp. 1198-1202 ◽  
Author(s):  
Nicholas J. Lynch ◽  
Silke Roscher ◽  
Thomas Hartung ◽  
Siegfried Morath ◽  
Misao Matsushita ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lipoteichoic Acid ◽  
Lectin Pathway ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Cell Wall Constituent ◽  
A Cell

Chemical analysis of isolated cell walls of Gram-positive bacteria and determination of the cell wall to cell mass ratio

1997 ◽  
Vol 28 (2) ◽  
pp. 147-157 ◽  
Author(s):  
Albert van der Wal ◽  
Willem Norde ◽  
Bernd Bendinger ◽  
Alexander J.B Zehnder ◽  
Johannes Lyklema
Keyword(s):  
Cell Wall ◽  
Chemical Analysis ◽  
Cell Walls ◽  
Cell Mass ◽  
Mass Ratio ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Isolated Cell

scholarly journals Methods for Confirming the Gram Reaction of Gram-variable Bacillus Species Isolated from Tobacco

2000 ◽  
Vol 19 (2) ◽  
pp. 97-101
Author(s):  
A Morin ◽  
N Poirier ◽  
S Vallee ◽  
A Porter
Keyword(s):  
Cell Wall ◽  
Bacillus Species ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Staining Techniques ◽  
Susceptibility Tests ◽  
Hydrolysis Of

AbstractBacillusis a predominant genus of bacteria isolated from tobacco. The Gram stain is the most commonly used and most important of all diagnostic staining techniques in microbiology. In order to help confirm the Gram positivity ofBacillusisolates from tobacco, three methods using the chemical differences of the cell wall and membrane of Gram-positive and Gram-negative bacteria were investigated: the KOH (potassium hydroxide), the LANA (L-alanine-4-nitroanilide), and the vancomycin susceptibility tests. When colonies of Gram-negative bacteria are treated with 3% KOH solution, a slimy suspension is produced, probably due to destruction of the cell wall and liberation of deoxyribonucleic acid (DNA). Gram-positive cell walls resist KOH treatment. The LANA test reveals the presence of a cell wall aminopeptidase that hydrolyzes the L-alanine-4-nitroanilide in Gram-negative bacteria. This enzyme is absent in Gram-positive bacteria. Vancomycin is a glycopeptide antibiotic inhibiting the cell wall peptido-glycan synthesis of Gram-positive microorganisms. Absence of lysis with KOH, absence of hydrolysis of LANA, and susceptibility to vancomycin were used with the Gram reaction to confirm the Gram positivity of variousBacillusspecies isolated from tobacco.B. laevolacticusexcepted, all Bacillus species tested showed negative reactions to KOH and LANA tests, and all species were susceptible to vancomycin (5 and 30 µg).

Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis

1989 ◽  
Vol 35 (8) ◽  
pp. 749-753 ◽  
Author(s):  
Denis Leclerc ◽  
Alain Asselin
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Gel Electrophoresis ◽  
Cell Walls ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Cell Wall Hydrolases

Cell walls from various Gram-positive bacteria were incorporated at a concentration of 0.2% (w/v) into polyacrylamide gels as a substrate for detection of cell wall hydrolases. Bacterial extracts from crude cell wall preparations were denatured with sodium dodecyl sulfate and 2-mercaptoethanol and subjected to denaturing polyacrylamide gel electrophoresis in gels containing bacterial cell walls. After renaturation in the presence of purified and buffered 1% (v/v) Triton X-100, cell wall hydrolases were visualized as clear lytic zones against the opaque cell wall background. One to fifteen bands with lytic activity could be detected, depending on bacterial extracts and on the nature of the cell walls incorporated into gels. Crude cell wall extracts were the best source of cell wall hydrolases from various Gram-positive bacteria such as Clostridium perfringens (15 bands), Micrococcus luteus (1 band), Bacillus megaterium (4 bands), Bacillus sp. (6 bands), B. cereus (3 bands), B. subtilis (7 bands), Staphylococcus aureus (13 bands), Streptococcus faecalis (3 bands), and Strep. pyogenes (5 bands). Molecular masses of cell wall hydrolases ranged from 17 to 114.6 kDa. Lytic activities against cell walls of Corynebacterium sepedonicum (Clavibacter michiganense pv. sepedonicum) could be shown with the cell wall extracts of Strep. pyogenes (45.7 kDa), Strep. faecalis (67 kDa), B. megaterium (67 kDa), and Staph. aureus (67 kDa).Key words: autolysins, electrophoresis, hydrolases, muramidases, peptidoglycan.

Microbial community analysis of a full-scale membrane bioreactor treating industrial wastewater

2008 ◽  
Vol 58 (8) ◽  
pp. 1589-1594 ◽  
Author(s):  
D. Naidoo ◽  
N. Ramdhani ◽  
F. Bux
Keyword(s):  
In Situ Hybridization ◽  
Microbial Community ◽  
Manufacturing Industry ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Positive Hybridization ◽  
Gram Negative Organisms

A Kubota™ submerged membrane bio-reactor was applied to treat wastewater from a sugar manufacturing industry. To achieve optimal results, fundamental and extended understanding of the microbiology is important. Fluorescence in situ hybridization was used to evaluate the microbial community present. The majority of cells visualized in the sludge flocs by staining with the DNA fluorochrome DAPI, hybridized strongly with a bacterial probe. Probes specific for the alpha-, beta-, and gamma-subclasses of proteobacteria and high G + C Gram positive bacteria were used to characterize the community structures by in situ hybridization. Sampling was carried out over 12 weeks and samples were fixed with 4% paraformaldehyde for gram positive organisms and ice cold ethanol for gram negative organisms. The activated sludge population usually constitutes about 80 to 90% of proteobacteria. However, in this study it was found that a relatively small amount of proteobacteria was present within the system. No positive hybridization signal was observed with any of the applied eubacterial family- level probes.

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