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 metalresponsive 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.This work has been supported by the Spanish Government, grant BFU2016-75425-P to Aurelio Moraleda-Muñoz (70% funded by FEDER)

Development versus predation: Transcriptomic changes during the lifecycle of Myxococcus xanthus

Myxococcus xanthus is a multicellular bacterium with a complex lifecycle. It is a soil-dwelling predator that preys on a wide variety of microorganisms by using a group and collaborative epibiotic strategy. In the absence of nutrients this myxobacterium enters in a unique developmental program by using sophisticated and complex regulatory systems where more than 1,400 genes are transcriptional regulated to guide the community to aggregate into macroscopic fruiting bodies filled of environmentally resistant myxospores. Herein, we analyze the predatosome of M. xanthus, that is, the transcriptomic changes that the predator undergoes when encounters a prey. This study has been carried out using as a prey Sinorhizobium meliloti, a nitrogen fixing bacteria very important for the fertility of soils. The transcriptional changes include upregulation of genes that help the cells to detect, kill, lyse, and consume the prey, but also downregulation of genes not required for the predatory process. Our results have shown that, as expected, many genes encoding hydrolytic enzymes and enzymes involved in biosynthesis of secondary metabolites increase their expression levels. Moreover, it has been found that the predator modifies its lipid composition and overproduces siderophores to take up iron. Comparison with developmental transcriptome reveals that M. xanthus downregulates the expression of a significant number of genes coding for regulatory elements, many of which have been demonstrated to be key elements during development. This study shows for the first time a global view of the M. xanthus lifecycle from a transcriptome perspective.Grant PID2020-112634GB-I0 funded by MCIN/AEI/ 10.13039/501100011033FEDER funds (grant A-BIO-126-UGR20

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 sigma2 domain and the sigma4.2 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.Spanish Government [CSD2009-00006, BFU2012-33248, 70% funded by FEDER]. Funding for open access charge: Grupo BIO318, Junta de Andalucía

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.Spanish Government (PID2020-112634GB-I00)FEDER funds (grant A-BIO-126-UGR20

Transcriptomic response of Sinorhizobium meliloti to the predatory attack of Myxococcus xanthus

Bacterial predation impacts microbial community structures, which can have both positive and negative effects on plant and animal health and on environmental sustainability. Myxococcus xanthus is an epibiotic soil predator with a broad range of prey, including Sinorhizobium meliloti, which establishes nitrogen-fixing symbiosis with legumes. During the M. xanthus-S. meliloti interaction, the predator must adapt its transcriptome to kill and lyse the target (predatosome), and the prey must orchestrate a transcriptional response (defensome) to protect itself against the biotic stress caused by the predatory attack. Here, we describe the transcriptional changes taking place in S. meliloti in response to myxobacterial predation. The results indicate that the predator induces massive changes in the prey transcriptome with up-regulation of protein synthesis and secretion, energy generation, and fatty acid (FA) synthesis, while down-regulating genes required for FA degradation and carbohydrate transport and metabolism. The reconstruction of up-regulated pathways suggests that S. meliloti modifies the cell envelop by increasing the production of different surface polysaccharides (SPSs) and membrane lipids. Besides the barrier role of SPSs, additional mechanisms involving the activity of efflux pumps and the peptide uptake transporter BacA, together with the production of H2O2 and formaldehyde have been unveiled. Also, the induction of the iron-uptake machinery in both predator and prey reflects a strong competition for this metal. With this research we complete the characterization of the complex transcriptional changes that occur during the M. xanthus-S. meliloti interaction, which can impact the establishment of beneficial symbiosis with legumes

Detoxification of azo dyes by a novel pH-versatile, salt-resistant laccase from Streptomyces ipomoea

A newly identified extracellular laccase produced by Streptomyces ipomoea CECT 3341 (SilA) was cloned and overexpressed, and its physicochemical characteristics assessed together with its capability to decolorize and detoxify an azotype dye. Molecular analysis of the deduced sequence revealed that SilA contains a TAT-type signal peptide at the N-terminus and only two cupredoxine domains; this is consistent with reports describing two other Streptomyces laccases but contrasts with most laccases, which contain three cupredoxine domains. The heterologous expression and purification of SilA revealed that the homodimer is the only active form of the enzyme. Its stability at high pH and temperature, together with its resistance to high concentrations of NaCl and to typical laccase inhibitors such as sodium azide confirmed the unique properties of this novel laccase. The range of substrates that SilA is able to oxidize was found to be pH-dependent; at alkaline pH, SilA oxidized a wide range of phenolic compounds, including the syringyl and guayacil moieties derived from lignin. The oxidative potential of this enzyme to use phenolic compounds as natural redox mediators was shown through the coordinated action of SilA and acetosyringone (as mediator), which resulted in the complete detoxification of the azo-type dye Orange II.This work was financially supported by the Spanish Ministry of Science (CICYT Project CTQ2004-0344/PPQ to MEA and BFU 2006-00972/BMC to JMD). We thank the Autonomous Government of Madrid and the University of Alcalá for the fellowship awarded to R.M

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