Generating a Collection of Insertion Mutations in the Staphylococcus aureus Genome Using bursa aurealis

Combination of dynamic turbidimetry and tube agglutination to identify procoagulant genes by transposon mutagenesis in Staphylococcus aureus

10.1101/426783 ◽

2018 ◽

Author(s):

Dong Luo ◽

Qiang Chen ◽

Bei Jiang ◽

Shirong Lin ◽

Linfeng Peng ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Protein Synthesis ◽

Energy Metabolism ◽

Gene Knockout ◽

Regulatory System ◽

Mutant Library ◽

Rt Pcr ◽

Pcr Techniques ◽

Insertion Mutations ◽

Insertion Mutants

Agglutinating function is responsible for an important pathogenic pattern in S.aureus. Although the mechanism of aggregation has been widely studied since S.aureus has been found, the agglutinating detailed process remains largely unknown. Here, we screened a transposon mutant library of Newman strain using tube agglutination and dynamic turbidmetry test and identified 8 genes whose insertion mutations lead to a decrease in plasma agglomerate ability. These partial candidate genes were further confirmed by gene knockout and gene complement as well as RT-PCR techniques. these insertion mutants, including NWMN_0166, NWMN_0674, NWMN_0756, NWMN_0952, NWMN_1282, NWMN_1228, NWMN_1345 and NWMN_1319, which mapped into coagulase, clumping factor A, oxidative phosphorylation, energy metabolism, protein synthesis and regulatory system, suggesting that these genes may play an important role in aggregating ability. The newly constructed knockout strains of coa, cydA and their complemented strains were also tested aggregating ability. The result of plasma agglutination was consistent between coa knockout strain and coa mutant strain, meanwhile, cydA complement strain didn’t restored its function. Further studies need to confirm these results. These findings provide novel insights into the mechanisms of aggregating ability and offer new targets for development of drugs in S.aureus.

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Cytochemical Studies of Cell Wall Biosynthesis in Staphylococcus Aureus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094905 ◽

1972 ◽

Vol 30 ◽

pp. 328-329

Author(s):

Masaatsu Koike ◽

Koichi Nakashima ◽

Kyoko Iida

Keyword(s):

Staphylococcus Aureus ◽

Periodate Oxidation ◽

Early Time ◽

The Other ◽

Penicillin G ◽

Cell Wall Biosynthesis ◽

Septal Wall ◽

Peptidoglycan Biosynthesis ◽

Gram Positive Bacteria ◽

Cross Linkage

Penicillin exerts the activity to inhibit the peptide cross linkage between each polysaccharide backbone at the final stage of wall-peptidoglycan biosynthesis of bacteria. Morphologically, alterations of the septal wall and mesosome in gram-positive bacteria, which were occurred in early time after treatment with penicillin, have been observed. In this experiment, these alterations were cytochemically investigated by means of silver-methenamine staining after periodate oxidation, which is applied for detection of localization of wall mucopolysaccharide.Staphylococcus aureus strain 209P treated with 100 u/ml of penicillin G was divided into two aliquotes. One was fixed by Kellenberger-Ryter's OSO4 fixative at 30, 60 and 120 min after addition of the antibiotic, dehydrated through alcohol series, and embedded in Epon 812 (Specimen A). The other was fixed by 21 glutaraldehyde, dehydrated through glycolmethacrylate series and embedded in glycolmethacrylate mixture, according to Bernhard's method (Specimen B).

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Gold labeling of teichoic acid on adjacent surfaces of staphylococcus aureus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160443 ◽

1990 ◽

Vol 48 (3) ◽

pp. 578-579

Author(s):

Margaret Hukee

Keyword(s):

Staphylococcus Aureus ◽

Teichoic Acid ◽

Protein Labeling ◽

Minimal Amount ◽

Section Thickness ◽

Double Labeling ◽

Parallel Surfaces ◽

Supporting Membrane

Gold labeling of two antigens (double labeling) is often done on two section surfaces separated by section thickness. Whether labeling is done on both sides of the same section or on two parallel surfaces separated by section thickness (PSSST), comparable results are dependent on an equal number of epitopes being exposed at each surface. We propose a method to study protein labeling within the same field of proteins, by examining two directly adjacent surfaces that were split during sectioning. The number of labeling sites on adjacent surfaces (AS) were compared to sites on PSSST surfaces in individual bacteria.Since each bacteria needed to be recognizable in all three section surfaces, one-hole grids were used for labeling. One-hole grids require a supporting membrane and excessive handling during labeling often ruptures the membrane. To minimize handling, a labeling chamber was designed that is inexpensive, disposable, minimizes contamination, and uses a minimal amount of solution.

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