Protein Complexes in Bacteria

Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometrycharacterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies

EVOLUTION OF PROTEIN COMPLEXES IN BACTERIAL SPECIES

Protein complexes are composed of two or more associated polypeptide chains that may have different functions. Protein complexes play a critical role for all processes in life and are considered as highly conserved in evolution. In previous studies, protein complexes from E. coli or Mycoplasma pneumoniae have been characterized experimentally, revealing that a typical bacterial cell has on the order of 500 protein complexes. Using gene homology (orthology), these experimentally-observed complexes can be used to predict protein complexes across many species of bacteria. Surprisingly, the majority of protein complexes is not conserved, demonstrating an unexpected evolutionary flexibility. The current research investigates the evolution of 174 well-characterized (“reference”) protein complexes from E. coli that have three or more subunits. More specifically, we study the evolutionary flexibility by using evidence and patterns of the presence or absence of the subunits across a range of 894 bacterial species and to interpret whether the evolution is due to the loss or gain of a subunit in the protein complex. The purpose of this study is to determine how the presence or absence of a subunit affects the protein complexes’ functionality. We discuss the functional changes observed in a protein complex due to the presence or absence of a particular subunit by using a statistical approach and by confirming its significance.https://scholarscompass.vcu.edu/uresposters/1253/thumbnail.jp

Misincorporation by RNA polymerase is a major source of transcription pausingin vivo

The transcription error rate estimated from mistakes in end product RNAs is 10−3–10−5. We analyzed the fidelity of nascent RNAs from all actively transcribing elongation complexes (ECs) in Escherichia coli and Saccharomyces cerevisiae and found that 1–3% of all ECs in wild-type cells, and 5–7% of all ECs in cells lacking proofreading factors are, in fact, misincorporated complexes. With the exception of a number of sequence-dependent hotspots, most misincorporations are distributed relatively randomly. Misincorporation at hotspots does not appear to be stimulated by pausing. Since misincorporation leads to a strong pause of transcription due to backtracking, our findings indicate that misincorporation could be a major source of transcriptional pausing and lead to conflicts with other RNA polymerases and replication in bacteria and eukaryotes. This observation implies that physical resolution of misincorporated complexes may be the main function of the proofreading factors Gre and TFIIS. Although misincorporation mechanisms between bacteria and eukaryotes appear to be conserved, the results suggest the existence of a bacteria-specific mechanism(s) for reducing misincorporation in protein-coding regions. The links between transcription fidelity, human disease, and phenotypic variability in genetically-identical cells can be explained by the accumulation of misincorporated complexes, rather than mistakes in mature RNA

Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function

Metabolic protein interactions in Bacillus subtilis studied at the single cell level

We have investigated protein-protein interactions in live Bacillus subtilis cells (a bacterium). B. subtilis’ natural habitat is the soil and the roots of plants, but also the human microbiota. B. subtilis is used worldwide as a model organism. Unlike eukaryotic cells, bacteria do not have organelles and therefore it was thought that the interior of bacteria is an unorganized mess. But bacteria do have a ‘skelet’: the cytoskeleton. More and more is known about the organization inside bacteria and it turns out that many proteins have a specific location in the cell. Protein complexes might be the bacterial equivalent of eukaryotic organelles. Earlier research suggests that protein complexes have a transient nature; they are formed when necessary and fall apart afterwards. During this research we studied the dynamics of a few protein complexes; of some proteins involved in sugar metabolism. This has been studied here in live bacteria with a miroscope. The method that we used to study protein-protein interactions is called FRET (Förster Resonance Energy Transfer). The principle of FRET can be explained with two stem forks. When one stem fork is excited it starts to vibrate; thereby spreading a wave. A second stem fork can absorb this wave if it is nearby and will start to vibrate with the same frequency. We have labeled sugar metabolism proteins with green and red fluorescent proteins (‘our two stem forks’). If we can observe the red protein after exciting the green protein, then we have proven that the sugar metabolism proteins interact

Hot and crispy : CRISPR-Cas systems in the hyperthermophile Sulfolobus solfataricus

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) genes are widely spread in bacteria and archaea, representing an intracellular defence system against invading viruses and plasmids. In the system, fragments from foreign DNA are captured and integrated into the host genome at the CRISPR locus. The locus is transcribed and the resulting RNAs are processed by Cas6 into small crRNAs (CRISPR RNAs) that guide a variety of effector complexes to degrade the invading genetic elements. Many bacteria and archaea have one major type of effector complex. However, Sulfolobus solfataricus strain P2 has six CRISPR loci with two families of repeats, four cas6 genes and three different types of effector complex. These features make S. solfataricus an important model for studying CRISPR-Cas systems. In the present article, we review our current understanding of crRNA biogenesis and its effector complexes, subtype I-A and subtype III-B, in S. solfataricus. We also discuss the differences in terms of mechanisms between the subtype III-B systems in S. solfataricus and Pyrococcus furiosus.PostprintPeer reviewe

Identification of a CD4+ T cell-stimulating antigen of pathogenic bacteria by expression cloning.

Identifying the immunogenic proteins that elicit pathogen-specific T cell responses is key to rational vaccine design. While several approaches have succeeded in identifying major histocompatibility complex (MHC) class I bound peptides that stimulate CD8+ T cells, these approaches have been difficult to extend to peptides presented by MHC class II molecules that stimulate CD4+ T cells. We describe here a novel strategy for identifying CD4+ T cell-stimulating antigen genes. Using Listeria monocytogenes-specific, lacZ-inducible T cells as single-cell probes, we screened a Listeria monocytogenes genomic library as recombinant Escherichia coli that were fed to macrophages. The antigen gene was isolated from the E. coli clone that, when ingested by the macrophages, allowed generation of the appropriate peptide/MHC class II complex and T cell activation. We show that the antigenic peptide is derived from a previously unknown listeria gene product with characteristics of a membrane-bound protein