ABSTRACT
The goal of this work was to create a model for the long persistence of Mycoplasma gallisepticum in depleted medium and under low growth temperature followed by proteomic study of the model. Nanoforms and revertants for M. gallisepticum were obtained. Proteomic maps were produced for different stages of the formation of nanoforms and revertants. It is shown that proteins responsible for essential cellular processes of glycolysis, translation elongation, and DnaK chaperone involved in the stabilization of newly synthesized proteins are crucial for the reversion of M. gallisepticum to a vegetative form. Based on the current data, it is assumed that changes in the metabolism of M. gallisepticum during nanoforming are not post-mortal, thus M. gallisepticum does not transform to uncultivable form, but remains in a reversible dormant state during prolonged unfavorable conditions.
Subject(s)
Bacterial Proteins/metabolism , Mycoplasma gallisepticum/metabolism , Proteome/metabolism , Bacterial Proteins/genetics , Mycoplasma gallisepticum/genetics , Proteome/genetics , Proteomics/methodsABSTRACT
Intact chloroplasts were prepared from protoplasts of the moss Physcomitrella patens according to an especially developed method. They were additionally separated into stroma and thylakoid fractions. The proteomes of intact plastids, stroma, and thylakoids were analyzed by 1D-electrophoresis under denaturing conditions followed by protein digestion and nano-LC-ESI-MS/MS of tryptic peptides from gel bands. A total of 624 unique proteins were identified, 434 of which were annotated as chloroplast resident proteins. The majority of proteins belonged to a photosynthetic group (21.3%) and to the group of proteins implicated in protein degradation, posttranslational modification, folding, and import (20.6%). Among proteins assigned to chloroplasts, the following groups are prominent combining proteins implicated in metabolism of: amino acids (6.9%), nucleotides (2.5%), lipids (2.2%), carbohydrates (2.4%), hormones (1.5%), isoprenoids (1.25%), vitamins and cofactors (1%), sulfur (1.25%), and nitrogen (1%); as well as proteins involved in the pentose-phosphate cycle (1.75%), tetrapyrrole synthesis (3.7%), and redox processes (3.6%). The data can be used in physiological and photobiological studies as well as in further studies of P. patens chloroplast proteome including structural and functional specifics of plant protein localization in organelles.
Subject(s)
Bryopsida/chemistry , Plant Proteins/chemistry , Proteome/chemistry , Chloroplasts/chemistry , Databases, Protein , Metabolic Networks and Pathways , Models, Biological , Protoplasts/chemistry , Subcellular Fractions/chemistryABSTRACT
The sequencing of the moss Physcomitrella patens genome has facilitated studies of the plant proteome. To develop a proteome reference map based on the genome sequence, we conducted 2D electrophoreses of proteins extracted from moss protoplasts, protonemata, and gametophores grown under standard conditions on Petri dishes. On silver-stained gels, depending on the developmental stage of the moss, we resolved from 500 to 600 protein spots that were then excised and digested by trypsin, and 212 proteins were identified by PMF-MALDI-TOF. To enhance the proteome coverage, we performed 1D SDS-PAGE with subsequent separation of tryptic peptides derived from digested gel band slices by LC-ESI-MS/MS. The proposed approach allowed us to identify 186 proteins had not been determined by 2D PMF-MALDI-TOF. Proteins identified by both methods were categorized using a system of clusterization of orthologous genes as metabolism (26%), cellular processes and signaling (16%), and information storage and processing (7%). Proteome analysis by differential gel electrophoresis revealed moderate differences between filamentous protonemata and leafy shoots. Surprisingly, protoplasts isolated from protonema filaments displayed significant differences in protein composition compared with both protonemata and gametophores.
Subject(s)
Bryophyta/chemistry , Proteome/chemistry , Proteomics , Bryophyta/genetics , Bryophyta/metabolism , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Proteome/genetics , Proteome/metabolism , Signal TransductionABSTRACT
Using modern proteomic assays, we have identified the products of gene expression and posttranslational modifications of proteins of the bacterium Mycoplasma gallisepticum S6. Combinations of different technologies of protein separation by electrophoresis and mass-spectrometric analysis gave us a total of 446 proteins, i.e. 61% of the annotated proteins of this microorganism. The Pro-Q Diamond and Pro-Q Emerald dye technology was used for fluorescent detection of ten phosphoproteins and two glycoproteins. The acylation of proteins was studied by electrophoresis after in vivo labeling with different 14C-labeled fatty acids, followed by autoradiography. Sixteen acylated proteins were identified, with a quarter of them involved in plasma membrane construction and another quarter involved in cell energy metabolism.
