The molecular mechanisms of the bacterial iron sensor IdeR.

Life came to depend on iron as a cofactor for many essential enzymatic reactions. However, once the atmosphere was oxygenated, iron became both scarce and toxic. Therefore, complex mechanisms have evolved to scavenge iron from an environment in which it is poorly bioavailable, and to tightly regulate intracellular iron contents. In bacteria, this is typically accomplished with the help of one key regulator, an iron-sensing transcription factor. While Gram-negative bacteria and Gram-positive species with low guanine-cytosine (GC) content generally use Fur (ferric uptake regulator) proteins to regulate iron homeostasis, Gram-positive species with high GC content use the functional homolog IdeR (iron-dependent regulator). IdeR controls the expression of iron acquisition and storage genes, repressing the former, and activating the latter in an iron-dependent manner. In bacterial pathogens such as Corynebacterium diphtheriae and Mycobacterium tuberculosis, IdeR is also involved in virulence, whereas in non-pathogenic species such as Streptomyces, it regulates secondary metabolism as well. Although in recent years the focus of research on IdeR has shifted towards drug development, there is much left to learn about the molecular mechanisms of IdeR. Here, we summarize our current understanding of how this important bacterial transcriptional regulator represses and activates transcription, how it is allosterically activated by iron binding, and how it recognizes its DNA target sites, highlighting the open questions that remain to be addressed.

The bacterial iron sensor IdeR recognizes its DNA targets by indirect readout.

The iron-dependent regulator IdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. IdeR makes few direct contacts with DNA bases in its target sequence, and we show here that these contacts are not required for target recognition. The results of our structural and mutational studies support a model wherein IdeR mainly uses an indirect readout mechanism, identifying its targets via the sequence-dependent DNA backbone structure rather than through specific contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins.

Transcriptional Regulators Controlling Virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a pathogen capable of colonizing virtually every human tissue. The host colonization competence and versatility of this pathogen are powered by a wide array of virulence factors necessary in different steps of the infection process. This includes factors involved in bacterial motility and attachment, biofilm formation, the production and secretion of extracellular invasive enzymes and exotoxins, the production of toxic secondary metabolites, and the acquisition of iron. Expression of these virulence factors during infection is tightly regulated, which allows their production only when they are needed. This process optimizes host colonization and virulence. In this work, we review the intricate network of transcriptional regulators that control the expression of virulence factors in P. aeruginosa, including one- and two-component systems and σ factors. Because inhibition of virulence holds promise as a target for new antimicrobials, blocking the regulators that trigger the production of virulence determinants in P. aeruginosa is a promising strategy to fight this clinically relevant pathogen.

Mechanisms of Action of Non-Canonical ECF Sigma Factors.

Extracytoplasmic function (ECF) sigma factors are subunits of the RNA polymerase specialized in activating the transcription of a subset of genes responding to a specific environmental condition. The signal-transduction pathways where they participate can be activated by diverse mechanisms. The most common mechanism involves the action of a membrane-bound anti-sigma factor, which sequesters the ECF sigma factor, and releases it after the stimulus is sensed. However, despite most of these systems following this canonical regulation, there are many ECF sigma factors exhibiting a non-canonical regulatory mechanism. In this review, we aim to provide an updated and comprehensive view of the different activation mechanisms known for non-canonical ECF sigma factors, detailing their inclusion to the different phylogenetic groups and describing the mechanisms of regulation of some of their representative members such as EcfG from Rhodobacter sphaeroides, showing a partner-switch mechanism; EcfP from Vibrio parahaemolyticus, with a phosphorylation-dependent mechanism; or CorE from Myxococcus xanthus, regulated by a metal-sensing C-terminal extension.

Metal-responsive RNA polymerase extracytoplasmic function (ECF) sigma factors.

In order to survive, bacteria must adapt to multiple fluctuations in their environment, including coping with changes in metal concentrations. Many metals are essential for viability, since they act as cofactors of indispensable enzymes. But on the other hand, they are potentially toxic because they generate reactive oxygen species or displace other metals from proteins, turning them inactive. This dual effect of metals forces cells to maintain homeostasis using a variety of systems to import and export them. These systems are usually inducible, and their expression is regulated by metal sensors and signal-transduction mechanisms, one of which is mediated by extracytoplasmic function (ECF) sigma factors. In this review, we have focused on the metal-responsive ECF sigma factors, several of which are activated by iron depletion (FecI, FpvI and PvdS), while others are activated by excess of metals such as nickel and cobalt (CnrH), copper (CarQ and CorE) or cadmium and zinc (CorE2). We focus particularly on their physiological roles, mechanisms of action and signal transduction pathways.

In depth analysis of the mechanism of action of metal-dependent sigma factors: characterization of CorE2 from Myxococcus xanthus.

Extracytoplasmic function sigma factors represent the third pillar of signal-transduction mechanisms in bacteria. The variety of stimuli they recognize and mechanisms of action they use have allowed their classification into more than 50 groups. We have characterized CorE2 from Myxococcus xanthus, which belongs to group ECF44 and upregulates the expression of two genes when it is activated by cadmium and zinc. Sigma factors of this group contain a Cys-rich domain (CRD) at the C terminus which is essential for detecting metals. Point mutations at the six Cys residues of the CRD have revealed the contribution of each residue to CorE2 activity. Some of them are essential, while others are either dispensable or their mutations only slightly affect the activity of the protein. However, importantly, mutation of Cys174 completely shifts the specificity of CorE2 from cadmium to copper, indicating that the Cys arrangement of the CRD determines the metal specificity. Moreover, the conserved CxC motif located between the σ2 domain and the σ4.2 region has also been found to be essential for activity. The results presented here contribute to our understanding of the mechanism of action of metal-dependent sigma factors and help to define new common features of the members of this group of regulators.

Bacterial predation: 75 years and counting!

The first documented study on bacterial predation was carried out using myxobacteria three quarters of a century ago. Since then, many predatory strains, diverse hunting strategies, environmental consequences and potential applications have been reported by groups all over the world. Now we know that predatory bacteria are distributed in a wide variety of environments and that interactions between predatory and non-predatory populations seem to be the most important factor in bacterial selection and mortality in some ecosystems. Bacterial predation has now been proposed as an evolutionary driving force. The structure and diversity of the predatory bacterial community is beginning to be recognized as an important factor in biodiversity due to its potential role in controlling and modelling bacterial populations in the environment. In this paper, we review the current understanding of bacterial predation, going over the strategies used by the main predatory bacteria to kill their prey. We have also reviewed and integrated the accumulated advances of the last 75 years with the interesting new insights that are provided by the analyses of genomes, predatomes, predatosomes and other comparative genomics studies, focusing on potential applications that derive from all of these areas of study.

Myxococcus xanthus predation: an updated overview.

Bacterial predators are widely distributed across a variety of natural environments. Understanding predatory interactions is of great importance since they play a defining role in shaping microbial communities in habitats such as soils. Myxococcus xanthus is a soil-dwelling bacterial predator that can prey on Gram-positive and Gram-negative bacteria and even on eukaryotic microorganisms. This model organism has been studied for many decades for its unusual lifecycle, characterized by the formation of multicellular fruiting bodies filled with myxospores. However, less is known about its predatory behavior despite being an integral part of its lifecycle. Predation in M. xanthus is a multifactorial process that involves several mechanisms working synergistically, including motility systems to efficiently track and hunt prey, and a combination of short-range and contact-dependent mechanisms to achieve prey death and feed on them. In the short-range attack, M. xanthus is best known for the collective production of secondary metabolites and hydrolytic enzymes to kill prey and degrade cellular components. On the other hand, contact-dependent killing is a cell-to-cell process that relies on Tad-like and type III secretion systems. Furthermore, recent research has revealed that metals also play an important role during predation, either by inducing oxidative stress in the prey, or by competing for essential metals. In this paper, we review the current knowledge about M. xanthus predation, focusing on the different mechanisms used to hunt, kill, and feed on its prey, considering the most recent discoveries and the transcriptomic data available.

Siderophores and competition for iron govern myxobacterial predation dynamics.

Bacterial predators are decisive organisms that shape microbial ecosystems. In this study, we investigated the role of iron and siderophores during the predatory interaction between two rhizosphere bacteria: Myxococcus xanthus, an epibiotic predator, and Sinorhizobium meliloti, a bacterium that establishes nitrogen-fixing symbiosis with legumes. The results show that iron enhances the motility of the predator and facilitates its predatory capability, and that intoxication by iron is not used by the predator to prey, although oxidative stress increases in both bacteria during predation. However, competition for iron plays an important role in the outcome of predatory interactions. Using combinations of predator and prey mutants (non-producers and overproducers of siderophores), we have investigated the importance of competition for iron in predation. The results demonstrate that the competitor that, via the production of siderophores, obtains sufficient iron for growth and depletes metal availability for the opponent will prevail in the interaction. Consequently, iron fluctuations in soils may modify the composition of microbial communities by altering the activity of myxobacterial predators. In addition, siderophore overproduction during predation can alter soil properties, affecting the productivity and sustainability of agricultural operations.

An ambruticin-sensing complex modulates Myxococcus xanthus development and mediates myxobacterial interspecies communication.

Starvation induces cell aggregation in the soil bacterium Myxococcus xanthus, followed by formation of fruiting bodies packed with myxospores. Sporulation in the absence of fruiting bodies can be artificially induced by high concentrations of glycerol through unclear mechanisms. Here, we show that a compound (ambruticin VS-3) produced by a different myxobacterium, Sorangium cellulosum, affects the development of M. xanthus in a similar manner. Both glycerol (at millimolar levels) and ambruticin VS-3 (at nanomolar concentrations) inhibit M. xanthus fruiting body formation under starvation, and induce sporulation in the presence of nutrients. The response is mediated in M. xanthus by three hybrid histidine kinases (AskA, AskB, AskC) that form complexes interacting with two major developmental regulators (MrpC, FruA). In addition, AskB binds directly to the mrpC promoter in vitro. Thus, our work indicates that the AskABC-dependent regulatory pathway mediates the responses to ambruticin VS-3 and glycerol. We hypothesize that production of ambruticin VS-3 may allow S. sorangium to outcompete M. xanthus under both starvation and growth conditions in soil.

The antibiotic crisis: How bacterial predators can help.

Discovery of antimicrobials in the past century represented one of the most important advances in public health. Unfortunately, the massive use of these compounds in medicine and other human activities has promoted the selection of pathogens that are resistant to one or several antibiotics. The current antibiotic crisis is creating an urgent need for research into new biological weapons with the ability to kill these superbugs. Although a proper solution requires this problem to be addressed in a variety of ways, the use of bacterial predators is emerging as an excellent strategy, especially when used as whole cell therapeutic agents, as a source of new antimicrobial agents by awakening silent metabolic pathways in axenic cultures, or as biocontrol agents. Moreover, studies on their prey are uncovering mechanisms of resistance that can be shared by pathogens, representing new targets for novel antimicrobial agents. In this review we discuss potential of the studies on predator-prey interaction to provide alternative solutions to the problem of antibiotic resistance.

Transcriptome dynamics of the Myxococcus xanthus multicellular developmental program.

The bacterium Myxococcus xanthus exhibits a complex multicellular life cycle. In the presence of nutrients, cells prey cooperatively. Upon starvation, they enter a developmental cycle wherein cells aggregate to produce macroscopic fruiting bodies filled with resistant myxospores. We used RNA-Seq technology to examine the transcriptome of the 96 hr developmental program. These data revealed that 1415 genes were sequentially expressed in 10 discrete modules, with expression peaking during aggregation, in the transition from aggregation to sporulation, or during sporulation. Analysis of genes expressed at each specific time point provided insights as to how starving cells obtain energy and precursors necessary for assembly of fruiting bodies and into developmental production of secondary metabolites. This study offers the first global view of developmental transcriptional profiles and provides important tools and resources for future studies.

Dissection of the sensor domain of the copper-responsive histidine kinase CorS from Myxococcus xanthus.

Myxococcus xanthus CorSR is a two-component system responsible for maintaining the response of this bacterium to copper. In the presence of this metal it upregulates, among others, the genes encoding the multicopper oxidase CuoA and the P1B -ATPase CopA. Dissection of the periplasmic sensor domain of the histidine kinase CorS by the analysis of a series of in-frame deletion mutants generated in this portion of the protein has revealed that copper sensing requires a region of 28 residues in the N terminus and another region of nine residues in the C terminus. Point mutations at His34, His38 and His171 demonstrate that they are essential for the ability of CorS to sense copper. Furthermore, the use of a bacterial two-hybrid system has revealed dimerization between monomers of CorS even in the absence of any metal, and that copper enhances this interaction. When dimerization was tested with proteins mutated at the three essential His residues, it was observed that these proteins maintain the intrinsic dimerization ability in the absence of metal. In contrast to the wild-type protein, copper did not strengthen the interaction, corroborating that copper binding to the three His residues of CorS is required for enhancing dimerization and transmitting the signal.