a b s t r a c tThe correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.

The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.

To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and 13C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.

Determination of the average cellular copy number of 400 proteins under different growth conditions and integration with protein turnover and absolute mRNA levels reveals the dynamics of protein expression in the genome-reduced bacterium Mycoplasma pneumoniae.

The effect of kinase, phosphatase and N-acetyltransferase deletions on proteome phosphorylation and acetylation was investigated in Mycoplasma pneumoniae. Bi-directional cross-talk between post-transcriptional modifications suggests an underlying regulatory molecular code in prokaryotes.

A new genome-scale metabolic reconstruction of M. pneumonia is used in combination with external metabolite measurement and protein abundance measurements to quantitatively explore the energy metabolism of this genome-reduce human pathogen.

Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.