Promiscuity and preferences of metallothioneins: the cell rules

Metalloproteins are essential for many cellular functions, but it has not been clear how they distinguish between the different metals to bind the correct ones. A report in BMC Biology finds that preferences of two metallothionein isoforms for two different cations are due to inherent properties of these usually less discriminating proteins. Here these observations are discussed in the context of the cellular mechanisms that regulate metal binding to proteins

Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group

<p>Abstract</p> <p>Background</p> <p>The <it>Bacillus cereus </it><it>sensu lato </it>group consists of six species (<it>B. anthracis</it>, <it>B. cereus</it>, <it>B. mycoides</it>, <it>B. pseudomycoides</it>, <it>B. thuringiensis</it>, and <it>B. weihenstephanensis</it>). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the <it>Bc </it>species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of <it>B. subtilis</it>.</p> <p>Results</p> <p>Phylogenetic analysis of the <it>Bc </it>species-group utilizing 157 single-copy genes of the family <it>Bacillaceae </it>suggests that several taxonomic revisions of the genus <it>Bacillus </it>should be considered. Within the <it>Bc </it>species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the <it>Bc </it>species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the <it>Bc </it>species-group.</p> <p>Conclusions</p> <p>Expansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in <it>B. subtilis</it>. Divergence of the sigma-controlled transcriptional regulons among various members of the <it>Bc </it>species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the <it>Bc </it>species-group.</p

Definition of the σW regulon of Bacillus subtilis in the absence of stress

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σW regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σX, σY, and σM regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σW-regulated. Under these conditions, σW exhibits a basal level of activity. Subsequently, we verified the σW-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σW anti-sigma factor RsiW and subsequent activation of the σW-regulon. Taken together, our studies identify 89 genes as being strictly σW-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σW-dependent genes were relatively mild, which implies that σW-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σW, but that this membrane protease also exerts other important post-transcriptional regulatory functions

The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core

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 σ 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-σ 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 σ R-binding residues are sequestered back into its hydrophobic core, releasing σ R to activate transcription of anti-oxidant genes

Promoter prediction and annotation of microbial genomes based on DNA sequence and structural responses to superhelical stress

BACKGROUND: In our previous studies, we found that the sites in prokaryotic genomes which are most susceptible to duplex destabilization under the negative superhelical stresses that occur in vivo are statistically highly significantly associated with intergenic regions that are known or inferred to contain promoters. In this report we investigate how this structural property, either alone or together with other structural and sequence attributes, may be used to search prokaryotic genomes for promoters. RESULTS: We show that the propensity for stress-induced DNA duplex destabilization (SIDD) is closely associated with specific promoter regions. The extent of destabilization in promoter-containing regions is found to be bimodally distributed. When compared with DNA curvature, deformability, thermostability or sequence motif scores within the -10 region, SIDD is found to be the most informative DNA property regarding promoter locations in the E. coli K12 genome. SIDD properties alone perform better at detecting promoter regions than other programs trained on this genome. Because this approach has a very low false positive rate, it can be used to predict with high confidence the subset of promoters that are strongly destabilized. When SIDD properties are combined with -10 motif scores in a linear classification function, they predict promoter regions with better than 80% accuracy. When these methods were tested with promoter and non-promoter sequences from Bacillus subtilis, they achieved similar or higher accuracies. We also present a strictly SIDD-based predictor for annotating promoter sequences in complete microbial genomes. CONCLUSION: In this report we show that the propensity to undergo stress-induced duplex destabilization (SIDD) is a distinctive structural attribute of many prokaryotic promoter sequences. We have developed methods to identify promoter sequences in prokaryotic genomes that use SIDD either as a sole predictor or in combination with other DNA structural and sequence properties. Although these methods cannot predict all the promoter-containing regions in a genome, they do find large sets of potential regions that have high probabilities of being true positives. This approach could be especially valuable for annotating those genomes about which there is limited experimental data

Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function

Disulfide relays and phosphorylative cascades: Partners in redox-mediated signaling pathways

Modifications of specific amino-acid residues of proteins are fundamental in order to modulate different signaling processes among which the cascade of phosphorylation represents the most effective example. Recently, also, the modification of the redox state of cysteine residues of certain proteins, which is a widespread mechanism in the regulation of protein function, has been proposed to be involved in signaling pathways. Growing evidence shows that some transcription factors could be modulated by both oxidation and phosphorylation. In particular, the pathways regulated by the mitogen activated protein (MAP) kinases represent well-established examples of the cross talk between redox-mediated signaling and phosphorylative cascades. This review will compare the two modes of signal transduction and propose an evolutionary model of a partnership of the two mechanisms in the eukaryotic cell, with redox-mediated signals being more specific and ancestral and phosphorylative signals being more diffuse but predominant in signal propagation. © 2005 Nature Publishing Group All rights reserved

Local Renyi entropic profiles of DNA sequences

<p>Abstract</p> <p>Background</p> <p>In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf) using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM). Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs.</p> <p>Results</p> <p>The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at <url>http://kdbio.inesc-id.pt/~svinga/ep/</url>.</p> <p>Conclusion</p> <p>The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures.</p

CorE from Myxococcus xanthus Is a Copper-Dependent RNA Polymerase Sigma Factor

The dual toxicity/essentiality of copper forces cells to maintain a tightly regulated homeostasis for this metal in all living organisms, from bacteria to humans. Consequently, many genes have previously been reported to participate in copper detoxification in bacteria. Myxococcus xanthus, a prokaryote, encodes many proteins involved in copper homeostasis that are differentially regulated by this metal. A σ factor of the ECF (extracytoplasmic function) family, CorE, has been found to regulate the expression of the multicopper oxidase cuoB, the P1B-type ATPases copA and copB, and a gene encoding a protein with a heavy-metal-associated domain. Characterization of CorE has revealed that it requires copper to bind DNA in vitro. Genes regulated by CorE exhibit a characteristic expression profile, with a peak at 2 h after copper addition. Expression rapidly decreases thereafter to basal levels, although the metal is still present in the medium, indicating that the activity of CorE is modulated by a process of activation and inactivation. The use of monovalent and divalent metals to mimic Cu(I) and Cu(II), respectively, and of additives that favor the formation of the two redox states of this metal, has revealed that CorE is activated by Cu(II) and inactivated by Cu(I). The activation/inactivation properties of CorE reside in a Cys-rich domain located at the C terminus of the protein. Point mutations at these residues have allowed the identification of several Cys involved in the activation and inactivation of CorE. Based on these data, along with comparative genomic studies, a new group of ECF σ factors is proposed, which not only clearly differs mechanistically from the other σ factors so far characterized, but also from other metal regulators

Hacking into bacterial biofilms: a new therapeutic challenge

Microbiologists have extensively worked during the past decade on a particular phase of the bacterial cell cycle known as biofilm, in which single-celled individuals gather together to form a sedentary but dynamic community within a complex structure, displaying spatial and functional heterogeneity. In response to the perception of environmental signals by sensing systems, appropriate responses are triggered, leading to biofilm formation. This process involves various molecular systems that enable bacteria to identify appropriate surfaces on which to anchor themselves, to stick to those surfaces and to each other, to construct multicellular communities several hundreds of micrometers thick, and to detach from the community. The biofilm microbial community is a unique, highly competitive, and crowded environment facilitating microevolutionary processes and horizontal gene transfer between distantly related microorganisms. It is governed by social rules, based on the production and use of "public" goods, with actors and recipients. Biofilms constitute a unique shield against external aggressions, including drug treatment and immune reactions. Biofilm-associated infections in humans have therefore generated major problems for the diagnosis and treatment of diseases. Improvements in our understanding of biofilms have led to innovative research designed to interfere with this process