Regulation of plasmid-encoded isoprene metabolism in Rhodococcus, a representative of an important link in the global isoprene cycle.

Emissions of biogenic volatile organic compounds (VOCs) form an important part of the global carbon cycle, comprising a significant proportion of net ecosystem productivity. They impact atmospheric chemistry and contribute directly and indirectly to greenhouse gases. Isoprene, emitted largely from plants, comprises one third of total VOCs, yet in contrast to methane, which is released in similar quantities, we know little of its biodegradation.

Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.

Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications.

Predicting the strength of UP-elements and full-length E. coli σE promoters.

Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σ(E), an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro.

Unexpected stress-reducing effect of PhaP, a poly(3-hydroxybutyrate) granule-associated protein, in Escherichia coli.

Phasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinant Escherichia coli, revealed by an increase in the concentrations of several heat stress proteins.

Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon.

The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed.

Using DNA microarrays to assay part function.

In recent years, the capability of synthetic biology to design large genetic circuits has dramatically increased due to rapid advances in DNA synthesis technology and development of tools for large-scale assembly of DNA fragments. Large genetic circuits require more components (parts), especially regulators such as transcription factors, sigma factors, and viral RNA polymerases to provide increased regulatory capability, and also devices such as sensors, receivers, and signaling molecules. All these parts may have a potential impact upon the host that needs to be considered when designing and fabricating circuits.

Predicting strength and function for promoters of the Escherichia coli alternative sigma factor, sigmaE.

Sequenced bacterial genomes provide a wealth of information but little understanding of transcriptional regulatory circuits largely because accurate prediction of promoters is difficult. We examined two important issues for accurate promoter prediction: (1) the ability to predict promoter strength and (2) the sequence properties that distinguish between active and weak/inactive promoters. We addressed promoter prediction using natural core promoters recognized by the well-studied alternative sigma factor, Escherichia coli sigma(E), as a representative of group 4 sigmas, the largest sigma group.

Reduced capacity of alternative sigmas to melt promoters ensures stringent promoter recognition.

In bacteria, multiple sigmas direct RNA polymerase to distinct sets of promoters. Housekeeping sigmas direct transcription from thousands of promoters, whereas most alternative sigmas are more selective, recognizing more highly conserved promoter motifs. For sigma(32) and sigma(28), two Escherichia coli Group 3 sigmas, altering a few residues in Region 2.

Promoter strength properties of the complete sigma E regulon of Escherichia coli and Salmonella enterica.

The sigma(E)-directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria. sigma(E) is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the sigma(E) regulon is driven from a suite of approximately 60 sigma(E)-dependent promoters.

Dissection of recognition determinants of Escherichia coli sigma32 suggests a composite -10 region with an 'extended -10' motif and a core -10 element.

Sigma32 controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of sigma32 promoters is a long -10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of sigma32 and in vivo and in vitro assays, we identified promoter recognition determinants of this motif.

Mutational analysis of Escherichia coli sigma28 and its target promoters reveals recognition of a composite -10 region, comprised of an 'extended -10' motif and a core -10 element.

Sigma28 controls the expression of flagella-related genes and is the most widely distributed alternative sigma factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing feature of sigma28 promoters is a long -10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif.

Technical considerations in using DNA microarrays to define regulons.

Transcription is the major regulatory target of gene expression in bacteria, and is controlled by many regulatory proteins and RNAs. Microarrays are a powerful tool to study the regulation of transcription on a genomic scale. Here we describe the use of transcription profiling and ChIP-chip to study transcriptional regulation in bacteria.

Convergence of molecular, modeling, and systems approaches for an understanding of the Escherichia coli heat shock response.

The heat shock response (HSR) is a homeostatic response that maintains the proper protein-folding environment in the cell. This response is universal, and many of its components are well conserved from bacteria to humans. In this review, we focus on the regulation of one of the most well-characterized HSRs, that of Escherichia coli.

SigmaE regulates and is regulated by a small RNA in Escherichia coli.

RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion.

Hfq modulates the sigmaE-mediated envelope stress response and the sigma32-mediated cytoplasmic stress response in Escherichia coli.

Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli, including the sigma(S)-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters.

Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress.

The heat-shock response (HSR), a universal cellular response to heat, is crucial for cellular adaptation. In Escherichia coli, the HSR is mediated by the alternative sigma factor, sigma32. To determine its role, we used genome-wide expression analysis and promoter validation to identify genes directly regulated by sigma32 and screened ORF overexpression libraries to identify sigma32 inducers.

Conserved and variable functions of the sigmaE stress response in related genomes.

Bacteria often cope with environmental stress by inducing alternative sigma (sigma) factors, which direct RNA polymerase to specific promoters, thereby inducing a set of genes called a regulon to combat the stress. To understand the conserved and organism-specific functions of each sigma, it is necessary to be able to predict their promoters, so that their regulons can be followed across species. However, the variability of promoter sequences and motif spacing makes their prediction difficult.

Uses and pitfalls of microarrays for studying transcriptional regulation.

Microarrays provide a powerful new tool for understanding the regulation of gene expression in bacteria. Many recent publications have used microarrays for identifying regulon members and stimulons that describe the complex organismal responses to environmental perturbations. The use of bioinformatics to identify DNA binding sites of transcription factors greatly facilitates the interpretation of these experiments.

Architectural requirements for optimal activation by tandem CRP molecules at a class I CRP-dependent promoter.

The Escherichia coli cyclic AMP receptor protein (CRP) activates transcription at target promoters by interacting with the C-terminal domain of the RNA polymerase alpha subunit. We have constructed a set of promoters carrying tandem DNA sites for CRP with one site centred at position -61.5 and the other site located at different upstream positions.