The Myxococcus xanthus Two-Component System CorSR Regulates Expression of a Gene Cluster Involved in Maintaining Copper Tolerance during Growth and Development

Myxococcus xanthus is a soil-dwelling member of the δ–Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains.This work has been funded by the Spanish Government (grants CSD2009-00006 and BFU2012-33248, 70% funded by FEDER). This work was also supported by the National Institute of General Medical Science of the National Institutes of Health under award number R01GM095826 to LJS, and by the National Science Foundation under award number MCB0742976 to LJS. JMD and JP received a fellowship from Junta de Andalucía to do some work at University of Georgia

CorE from Myxococcus xanthus Is a Copper-Dependent RNA Polymerase Sigma Factor

The dual toxicity/essentiality of copper forces cells to maintain a tightly regulated homeostasis for this metal in all living organisms, from bacteria to humans. Consequently, many genes have previously been reported to participate in copper detoxification in bacteria. Myxococcus xanthus, a prokaryote, encodes many proteins involved in copper homeostasis that are differentially regulated by this metal. A σ factor of the ECF (extracytoplasmic function) family, CorE, has been found to regulate the expression of the multicopper oxidase cuoB, the P1B-type ATPases copA and copB, and a gene encoding a protein with a heavy-metal-associated domain. Characterization of CorE has revealed that it requires copper to bind DNA in vitro. Genes regulated by CorE exhibit a characteristic expression profile, with a peak at 2 h after copper addition. Expression rapidly decreases thereafter to basal levels, although the metal is still present in the medium, indicating that the activity of CorE is modulated by a process of activation and inactivation. The use of monovalent and divalent metals to mimic Cu(I) and Cu(II), respectively, and of additives that favor the formation of the two redox states of this metal, has revealed that CorE is activated by Cu(II) and inactivated by Cu(I). The activation/inactivation properties of CorE reside in a Cys-rich domain located at the C terminus of the protein. Point mutations at these residues have allowed the identification of several Cys involved in the activation and inactivation of CorE. Based on these data, along with comparative genomic studies, a new group of ECF σ factors is proposed, which not only clearly differs mechanistically from the other σ factors so far characterized, but also from other metal regulators

Caracterización molecular de la oxidasa multicobre CuoA DE Myxococcus xanthus

Tesis Univ. Granada. Departamento de Microbiología. Leída el 11 de diciembre de 200

Differential Expression of the Three Multicopper Oxidases from Myxococcus xanthus▿

Myxococcus xanthus is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact, M. xanthus vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells preadapted to this metal reach the same levels of resistance during both stages. Analysis of the M. xanthus genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of cuoA and cuoB respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of cuoC shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity

Expression of three developmental genes, <i>mrpC</i>, <i>fruA</i> and <i>tps</i>, in copper pre-adapted cells.

<p>WT (solid columns) or Δ<i>corSR</i> (dashed columns) cells harboring <i>mrpC-lacZ</i>, <i>fruA-lacZ</i> and <i>tps-lacZ</i> were grown in the presence of 600 µM copper, concentrated to an OD₆₀₀ of 15, and spotted onto CF medium containing the indicated copper concentrations. Cells were harvested at several time-points to determine specific β-galactosidase activity.</p

<i>In silico</i> analysis of the nine genes of the <i>curA</i> operon.

a<p>MXAN: gene identifiers in <i>M. xanthus</i> genome.</p>b<p>Predicted function inferred from BLASTP, Pfam, ScanProsite, MotifScan, and InterProScan. The Pfam identifiers are in parentheses.</p>c<p>Signal peptides (SP) were deduced from results at SignalP and SOSUI servers.</p>d<p>Transmembrane domains (TM) from TMHMM and SOSUI servers. The numbers of predicted transmembrane domains are indicated in parentheses.</p><p>Programs and databases used are available through ExPASy server (<a href="http://www.expasy.org/" target="_blank">http://www.expasy.org/</a>).</p

Carotenoid accumulation in different strains during growth and development.

<p><b>A.</b> WT, Δ<i>corSR</i>, and Δ<i>corSR</i> Δ<i>carB</i> strains were grown in the absence of copper, concentrated at an OD₆₀₀ of 15, and spotted onto CTT agar plates containing a copper gradient from 0 to 1000 µM. Pictures were taken after 48-h incubation. <b>B.</b> Carotenoid accumulation during development. In these experiments cells grown in the absence of copper were concentrated at an OD₆₀₀ of 15 and spotted onto CF media with 40 µM copper. Pictures were taken at 72 h. Bar represents 100 µm. <b>C.</b> Carotenoid accumulation in the Δ<i>3414</i> and the WT strains during growth in the presence of different copper concentrations. Cells were grown in the absence of copper, concentrated to an OD₆₀₀ of 15, and spotted onto CTT agar plates containing the indicated copper concentrations.</p

Growth of WT and Δ<i>corSR</i> strains in the presence of copper.

<p><b>A.</b> Cells grown in the absence of copper were diluted into CTT liquid media containing the indicated copper concentrations. <b>B.</b> Cells were adapted to grow in the presence of 600 µM of copper prior to dilution in CTT liquid media containing the indicated copper concentrations. OD₆₀₀ in both panels was monitored after 24-h incubation. Error bars indicate standard deviations. WT, blue lines; Δ<i>corSR,</i> red lines.</p

Measurements of intracellular copper accumulation during growth and development.

<p>Cells from WT, Δ<i>corSR,</i> and Δ<i>3414</i> strains were incubated on CTT (growth) or CF (development) agar plates containing the indicated copper concentrations for 72 h prior to harvesting for copper determinations.</p