ABSTRACT
Mycoplasma gallisepticum belongs to the class Mollicutes and induces severe chronic respiratory disease in chickens. It lacks the cell wall and contains a very small genome and, accordingly, a reduced set of regulatory proteins. It is assumed that one of the regulatory mechanisms in mycoplasmas may be the dynamics of the spatial organization of the chromosome. M. gallisepticum has only two known nucleoid-associated (NAP) histone-like proteins (Hup_1 and Hup_2). To search for new potential NAP that may play a role in the infection process, we isolated nucleoid fractions from M. gallisepticum cells before and after infection of HD3 chicken erythroblast cell line and performed a comparative proteomic analysis of these fractions. We identified several potential NAP that included the components of the terminal organelle and adhesion, VlhA antigen, NADH oxidase, and PykF pyruvate kinase.
Subject(s)
Mycoplasma Infections , Mycoplasma gallisepticum , Animals , Chickens , Mycoplasma gallisepticum/genetics , Mycoplasma gallisepticum/metabolism , Organelles , ProteomicsABSTRACT
It is assumed that unknown mechanisms can be involved in adaptation Mycoplasma gallisepticum to unfavorable factors, one of these can be local rearrangements of the structure and spatial organization of the chromosome. To study these mechanisms, we obtained a culture of M. gallisepticum with synchronized division and isolated the nucleoid fraction from this culture by the method of mild cell lysis and centrifugation in a sucrose gradient. Liquid chromatography-mass spectrometry analysis of the proteome showed that in comparison with the cell lysate, the nucleoid fraction was enriched with DNA-binding proteins. This analysis will help to find new nucleoid-associated proteins and to study their dynamics, distribution, and their role during infection and under stress conditions.
Subject(s)
Bacterial Proteins/isolation & purification , DNA-Binding Proteins/isolation & purification , Mycoplasma gallisepticum/genetics , Nuclear Proteins/isolation & purification , Proteome/isolation & purification , Bacterial Proteins/classification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Division , Centrifugation, Density Gradient/methods , Chromatography, Liquid , Culture Media/chemistry , DNA, Bacterial/genetics , DNA-Binding Proteins/classification , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression , Mass Spectrometry , Mycoplasma gallisepticum/metabolism , Nuclear Proteins/classification , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proteome/classification , Proteome/genetics , Proteome/metabolismABSTRACT
Culturing of Mycoplasma hominis in the presence of arginine and thymidine and subsequent comparative proteomic analysis of cells showed that, in addition to the already known arginine dihydrolase pathway of energy metabolism, M. hominis can utilize deoxyribose phosphates formed as a result of catabolism of pyrimidine nucleosides. In this case, a sharp deceleration of cell growth was observed. This allows M. hominis to occupy new niches in the host organism and survive under competitive conditions when the main sources of energy are unavailable.
Subject(s)
Carbon/pharmacology , Culture Media/pharmacology , Mycoplasma hominis/metabolism , Proteome/analysis , Arginine/pharmacology , Bacteriological Techniques/methods , Culture Media/chemistry , Electrophoresis, Gel, Two-Dimensional , Humans , Mycoplasma Infections/metabolism , Mycoplasma Infections/microbiology , Mycoplasma hominis/chemistry , Mycoplasma hominis/drug effects , Mycoplasma hominis/growth & development , Proteome/drug effects , Proteome/metabolism , Proteomics/methods , Thymidine/pharmacologyABSTRACT
Mycoplasma gallisepticum (M. gallisepticum) belongs to the class of Mollicutes. It causes chronic respiratory disease in avian species. It is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, M. gallisepticum has a limited variety of DNA-binding proteins (DBP) and transcription factors. Consequently, the diversity of DNA-binding proteins and transcription factors (TF) in M. gallisepticum is limited in comparison with related bacteria such as Bacillus subtilis. Studies have shown, however, that mycoplasmas demonstrate a wide range of differential expression of genes in response to various stress factors, which promotes effective adaptation to unfavorable conditions. We assume that in the case of mycoplasmas, which are characterized by a combination of the reduction of known gene expression regulation systems and a high adaptive potential, the coordination of gene expression can be provided due to local changes in the structure and spatial organization of the chromosome. The study of the dynamic changes of the proteomic profile of M. gallisepticum nucleoid may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation including its changes upon invasion of the host cells.
ABSTRACT
I(min) is a plasma membrane-located, Ca(2+)-selective channel that is activated by store depletion and regulated by inositol 1,4, 5-trisphosphate (IP(3)). In the present work we examined the coupling between I(min) and IP(3) receptors in excised plasma membrane patches from A431 cells. I(min) was recorded in cell-attached mode and the patches were excised into medium containing IP(3). In about 50% of experiments excision caused the loss of activation of I(min) by IP(3.) In the remaining patches activation of I(min) by IP(3) was lost upon extensive washes of the patch surface. The ability of IP(3) to activate I(min) was restored by treating the patches with rat cerebellar microsomes reach in IP(3) receptors but not by control forebrain microsomes. The re-activated I(min) had the same kinetic properties as I(min) when it is activated by Ca(2+)-mobilizing agonists in intact cells and by IP(3) in excised plasma membrane patches and it was inhibited by the I(crac) inhibitor SKF95365. We propose that I(min) is a form of I(crac) and is gated by IP(3) receptors.