HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium invades intestinal epithelial cells using a type three secretion system (TTSS) encoded on Salmonella Pathogenicity Island 1 (SPI1). The SPI1 TTSS injects effector proteins into the cytosol of host cells where they promote actin rearrangement and engulfment of the bacteria. We previously identified RtsA, an AraC-like protein similar to the known HilC and HilD regulatory proteins. Like HilC and HilD, RtsA activates expression of SPI1 genes by binding upstream of the master regulatory gene hilA to induce its expression. HilA activates the SPI1 TTSS structural genes. Here we present evidence that hilA expression, and hence the SPI1 TTSS, is controlled by a feedforward regulatory loop. We demonstrate that HilC, HilD and RtsA are each capable of independently inducing expression of the hilC, hilD and rtsA genes, and that each can independently activate hilA. Using competition assays in vivo, we show that each of the hilA regulators contribute to SPI1 induction in the intestine. Of the three, HilD has a predominant role, but apparently does not act alone either in vivo or in vitro to sufficiently activate SPI1. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through HilD, thus inducing hilC, rtsA and hilA. However, the two-component systems are not responsible for environmental regulation of SPI1. Rather, we show that 'SPI1 inducing conditions' cause independent activation of the rtsA, hilC and hilD genes in the absence of known regulators. Our model of SPI1 regulation provides a framework for future studies aimed at understanding this complicated regulatory network.     

An analysis of the class of gene regulatory functions implied by a biochemical model

Understanding the integrated behavior of genetic regulatory networks, in which genes regulate one another's activities via RNA and protein products, is emerging as a dominant problem in systems biology. One widely studied class of models of such networks includes genes whose expression values assume Boolean values (i.e., on or off). Design decisions in the development of Boolean network models of gene regulatory systems include the topology of the network (including the distribution of input- and output-connectivity) and the class of Boolean functions used by each gene (e.g., canalizing functions, post functions, etc.). For example, evidence from simulations suggests that biologically realistic dynamics can be produced by scale-free network topologies with canalizing Boolean functions. This work seeks further insights into the design of Boolean network models through the construction and analysis of a class of models that include more concrete biochemical mechanisms than the usual abstract model, including genes and gene products, dimerization, cis-binding sites, promoters and repressors. In this model, it is assumed that the system consists of N genes, with each gene producing one protein product. Proteins may form complexes such as dimers, trimers, etc. The model also includes cis-binding sites to which proteins may bind to form activators or repressors. Binding affinities are based on structural complementarity between proteins and binding sites, with molecular binding sites modeled by bit-strings. Biochemically plausible gene expression rules are used to derive a Boolean regulatory function for each gene in the system. The result is a network model in which both topological features and Boolean functions arise as emergent properties of the interactions of components at the biochemical level. A highly biased set of Boolean functions is observed in simulations of networks of various sizes, suggesting a new characterization of the subset of Boolean functions that are likely to appear in gene regulatory networks.     

A pair of highly conserved two-component systems participates in the regulation of the hypervariable FIR proteins in different Legionella species

Legionella pneumophila and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the Icm/Dot type IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which functions as its chaperone, are two essential components of this system. It was previously shown that in 29 Legionella species, a large hypervariable-gene family (fir genes) is located upstream from a conserved icmQ gene, but although nonhomologous, the FIR proteins were found to function similarly together with their corresponding IcmQ proteins. Alignment of the regulatory regions of 29 fir genes revealed that they can be divided into three regulatory groups; the first group contains a binding site for the CpxR response regulator, which was previously shown to regulate the L. pneumophila fir gene (icmR); the second group, which includes most of the fir genes, contains the CpxR binding site and an additional regulatory element that was identified here as a PmrA binding site; and the third group contains only the PmrA binding site. Analysis of the regulatory region of two fir genes, which included substitutions in the CpxR and PmrA consensus sequences, a controlled expression system, as well as examination of direct binding with mobility shift assays, revealed that both CpxR and PmrA positively regulate the expression of the fir genes that contain both regulatory elements. The change in the regulation of the fir genes that occurred during the course of evolution might be required for the adaptation of the different Legionella species to their specific environmental hosts.     

Characterization of genes with tissue-specific differential expression patterns in Populus

Like many plants, Populus has an evolutionary history in which several, both recent and more ancient, genome duplication events have occurred and, therefore, constitutes an excellent model system for studying the functional evolution of genes. In the present study, we have focused on the properties of genes with tissue-specific differential expression patterns in poplar. We identified the genes by analyzing digital expression profiles derived by mapping 90,000+ expressed sequence tags (ESTs) from 18 sources to the predicted genes of Populus. Our sequence analysis suggests that tissue-specific differentially expressed genes have less diverged paralogs than average, indicating that gene duplication events is an important event in the pathway leading to this type of expression pattern. The functional analysis showed that genes coding for proteins involved in processes of functional importance for the specific tissue(s) in which they are expressed and genes coding for regulatory or responsive proteins are most common among the differentially expressed genes, demonstrating that the expression differentiation process is under strong selective pressure. Thus, our data supports a model where gene duplication followed by gene specialization or expansion of the regulatory and responsive networks leads to tissue-specific differential expression patterns. We have also searched for clustering of genes with similar expression pattern into gene-expression neighborhoods within the Populus genome. However, we could not detect any major clustering among the analyzed genes with highly specific expression patterns. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.