Eberhardt, A., Wu, L., Errington, J., Vollmer, W.
The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.
Rajasekar, K., Zdanowski, K., Yan, J., Hopper, J.
Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor Ï(R) preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex. Oxidation of RsrA is limited by the rate of zinc release, which weakens the RsrA-Ï(R) complex by accelerating its dissociation. The subsequent trigger disulfide, formed between specific combinations of RsrA's three zinc-binding cysteines, precipitates structural collapse to a compact state where all Ï(R)-binding residues are sequestered back into its hydrophobic core, releasing Ï(R) to activate transcription of anti-oxidant genes.
Campbell, E., Tupy, J., Gruber, T., Wang, S.
The sigma factors are the key regulators of bacterial transcription. ECF (extracytoplasmic function) sigma's are the largest and most divergent group of sigma(70) family members. ECF sigma's are normally sequestered in an inactive complex by their specific anti-sigma factor, which often spans the inner membrane. Here, we determined the 2 A resolution crystal structure of the Escherichia coli ECF sigma factor sigma(E) in an inhibitory complex with the cytoplasmic domain of its anti-sigma, RseA. Despite extensive sequence variability, the two major domains of sigma(E) are virtually identical in structure to the corresponding domains of other sigma(70) family members. In combination with a model of the sigma(E) holoenzyme and biochemical data, the structure reveals that RseA functions by sterically occluding the two primary binding determinants on sigma(E) for core RNA polymerase.
Blatch, G., LÃ¤ssle, M.
The tetratricopeptide repeat (TPR) motif is a protein-protein interaction module found in multiple copies in a number of functionally different proteins that facilitates specific interactions with a partner protein(s). Three-dimensional structural data have shown that a TPR motif contains two antiparallel alpha-helices such that tandem arrays of TPR motifs generate a right-handed helical structure with an amphipathic channel that might accommodate the complementary region of a target protein. Most TPR-containing proteins are associated with multiprotein complexes, and there is extensive evidence indicating that TPR motifs are important to the functioning of chaperone, cell-cycle, transcription, and protein transport complexes. The TPR motif may represent an ancient protein-protein interaction module that has been recruited by different proteins and adapted for specific functions. BioEssays 1999;21:932-939.
Toyoda, K., Inui, M.
Bacteria modify their expression of different terminal oxidases in response to oxygen availability. Corynebacterium glutamicum, a facultative anaerobic bacterium of the phylum Actinobacteria, possesses aa3 -type cytochrome c oxidase and cytochrome bd-type quinol oxidase, the latter of which is induced by oxygen limitation. We report that an extracytoplasmic function Ï factor, Ï(C) , is responsible for the regulation of this process. Chromatin immunoprecipitation with microarray analysis detected eight Ï(C) -binding regions in the genome, facilitating the identification of a consensus promoter sequence for Ï(C) recognition. The promoter sequences were found upstream of genes for cytochrome bd, heme a synthesis enzymes and uncharacterized membrane proteins, all of which were upregulated by sigC overexpression. However, one consensus promoter sequence found on the antisense strand upstream of an operon encoding the cytochrome bc1 complex conferred a Ï(C) -dependent negative effect on expression of the operon. The Ï(C) regulon was induced by cytochrome aa3 deficiency without modifying sigC expression, but not by bc1 complex deficiency. These findings suggest that Ï(C) is activated in response to impaired electron transfer via cytochrome aa3 and not directly to a shift in oxygen levels. Our results reveal a new paradigm for transcriptional regulation of the aerobic respiratory system in bacteria.