Exploration of Modulated Genetic Circuits Governing Virulence Determinants in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The expression of virulence genes in the human pathogen Staphylococcus aureus is strongly influenced by the multiple global regulators. The signal transduction cascade of these global regulators is accountable for recognizing and integrating the environmental cues to regulate the virulence regulon. While the production of virulent factors by individual global regulators are comparatively straightforward to define, auto-regulation of these global regulators and their impact on other regulators is more complex process. There are several reports on the production of virulent factors that are precisely regulated by switching processes of multiple global regulators including some prominent accessory regulators such as agr, sae and sar which allows S. aureus to coordinate the gene expression, and thus, provide organism an ability to act collectively. This review implicates the mechanisms involved in the global regulation of various virulence factors along with a comprehensive discussion on the differences between these signal transduction systems, their auto-induction and, coordination of classical and some comparatively new bacterial signal transduction systems.     

Riboswitch regulation mechanisms: RNA,metabolites and regulatory proteins

Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.     

Regulation of bacteriocin production in Lactobacillus plantarum depends on a conserved promoter arrangement with consensus binding sequence

Bacteriocin production in Lactobacillus plantarum C11 is regulated by a three-component signal transduction system comprising a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two homologous response regulators (RRs; PlnC and PlnD). Both RRs are DNA-binding proteins that bind to promoter-proximal elements in the pln regulon. The binding site for the two regulators consists of two 9-bp direct repeats, that conform to the consensus sequence 5'-TACGTTAAT-3', and the repeats are separated by an intervening 12-bp AT-rich spacer region. In the present work, the plhA promoter was used as a model to evaluate the significance of the binding sequence and conserved promoter arrangement. Point substitutions in the consensus sequence, particularly those in invariant positions, either abolished or significantly reduced binding of PlnC and PlnD. Both regulators bind as homodimers to DNA fragments containing a complete set of regulatory elements, while removal of either repeat, or alterations in the length of the spacer region, significantly weakened binding of both protein dimers. DNase I footprinting demonstrated that PlnC and PlnD both bind to, and protect, the direct repeats. By fusing the plnA promoter region to the beta-glucuronidase (GUS) gene, it was shown that promoter activity is dependent on an intact set of accurately organized repeats. The in vitro and in vivo results presented here confirm the involvement of the repeats as regulatory elements in the regulation of bacteriocin production.     

Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.     

Statistical fluctuations in processes of gene expression regulation: consideration of the problem from point of view of statistical mechanics

The regulation of gene expression is a basic problem of biology. In some cases, the gene activity is regulated by specific binding of regulatory proteins to DNA. In terms of statistical mechanics, this binding is described as the process of adsorption of ligands on the one-dimensional lattice and has a probability nature. As a random physical process, the adsorption of regulatory proteins on DNA introduces a noise to the regulation of gene activity. We derived equations, which make it possible to estimate this noise in the case of the binding of the lac repressor to the operator and showed that these estimates correspond to experimental data. Many ligands are able to bind nonspecifically to DNA. Nonspecific binding is characterized by a lesser equilibrium constant but a greater number of binding sites on the DNA, as compared with specific binding. Relations are presented, which enable one to estimate the probability of the binding of a ligand on a specific site and on nonspecific sites on DNA. The competition between specific and nonspecific binding of regulatory proteins plays a great role in the regulation of gene activity. Similar to the one-dimensional "lattice gas" of particles, ligands adsorbed on DNA produce "one-dimensional" pressure on proteins located at the termini of free regions of DNA. This pressure, an analog of osmotic pressure, may be of importance in processes leading to changes in chromatin structure and activation of gene expression.     

Recruitment of host ATP-dependent proteases by bacteriophage lambda

Upon infection of a bacterial cell, the temperate bacteriophage lambda executes a regulated temporal program with two possible outcomes: (1) Cell lysis and virion production or (2) establishment of a dormant state, lysogeny, in which the phage genome (prophage) is integrated into the host chromosome. The prophage is replicated passively as part of the host chromosome until it is induced to resume the lytic cycle. In this review, we summarize the evidence that implicates every known ATP-dependent protease in the regulation of specific steps in the phage life cycle. The proteolysis of specific regulatory proteins appears to fine-tune phage gene expression. The bacteriophage utilizes multiple proteases to irreversibly inactivate specific regulators resulting in a temporally regulated program of gene expression. Evolutionary forces may have favored the utilization of overlapping protease specificities for differential proteolysis of phage regulators according to different phage life styles.     

The cAMP Receptor-Like Protein CLP Is a Novel c-di-GMP Receptor Linking Cell-Cell Signaling to Virulence Gene Expression in Xanthomonas campestris

Cyclic-di-GMP [bis-(3′-5′)-cyclic diguanosine monophosphate] controls a wide range of functions in eubacteria, yet little is known about the underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris, expression of a subset of virulence genes is regulated by c-di-GMP and also by the CAP (catabolite activation protein)-like protein XcCLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and functional insights into the interplay between XcCLP and c-di-GMP in regulation of gene expression. XcCLP bound target promoter DNA with submicromolar affinity in the absence of any ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to XcCLP with micromolar affinity. The crystal structure of XcCLP showed that the protein adopted an intrinsically active conformation for DNA binding. Alteration of residues of XcCLP implicated in c-di-GMP binding through modeling studies caused a substantial reduction in binding affinity for the nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by c-di-GMP. Together, these findings reveal the structural mechanism behind a novel class of c-di-GMP effector proteins in the CRP/FNR superfamily and indicate that XcCLP regulates bacterial virulence gene expression in a manner negatively controlled by the c-di-GMP concentrations.