Resumen de Análisis de la respuesta al cobre en myxococcus xanthus mediada por cuob y cuoc. Caracterización del factor sigma de tipo ecf core
Myxococcus xanthus is a ¿-Proteobacterium that glides on the soil, either as single cells or more typically as groups feeding cooperatively. However, under conditions of nutrient depletion, this bacterium undergoes a developmental program, unique among the prokaryotes in which cells come together in a coordinated way to produce multicellular fruiting bodies within they sporulate, converting the vegetative cells into myxospores. This aggregation and differentiation of bacteria requires a complex interaction of signals controlling gene expression in time and space, ability that has converted this ¿-Proteobacterium in a model for studying prokaryotic morphogenesis and differentiation.
M. xanthus is common in soils, rich in organic materials and rotting wood. In these niches, it has to frequently cope with toxic concentrations of copper ions, which is an essential trace element toxic in excess. The importance of maintaining copper homeostasis is further reinforced by the presence in the cells of several protein families known to control the intracellular concentration of metal ions or helping to confine them to vital roles. Moreover, in higher-order organisms, such as humans, disturbed copper homeostasis has been implicated in diseases such as Menkes and Wilson syndromes, Alzheimer's and Parkinson's diseases.
The copper response in M. xanthus seems to be more complex than in other bacteria. It has been reported that copper induces the accumulation of carotenoids in dark-grown cultures of M. xanthus, activating the transcription of the structural genes for carotenoid synthesis. Analysis of the M. xanthus genome has revealed a plethora of gene products with sequence similarities to proteins known to be involved in copper handling and trafficking in other organisms, most of which are redundant. This finding is indicative that copper homeostasis mechanisms in this bacterium could be different during growth and development. Among others, we have identified genes that encode three multicopper oxidases (MCOs) which have been named cuoA, cuoB, and cuoC (for cuprous oxidases).
The MCO family is defined by the presence of three spectroscopically different copper centers. Enzymes that belong to this family include laccases, ascorbate oxidases, and ceruloplasmin. Even though several bacterial MCOs have also been described, the biological roles of MCOs of prokaryotic origins are diverse and apparently unrelated. Some of them have been implicated in copper tolerance, and the coexistence in the M. xanthus genome of three paralogs for MCOs raises intriguing questions about their physiological roles in copper homeostasis along the life cycle.
The aim of this work was to study the role of two of these multicopper oxidases (CuoB and CuoC) in this bacterium, and the regulation of one of them by a novel ECF sigma factor (CorE).
CuoB is involved in the primary adaptive response to copper; hence, its promoter responds rapidly after copper addition, and its expression decreases as the expression of cuoC gene increases. As a result, ¿cuoB mutant cells are extremely sensitive to this metal. On the contrary, CuoC seems to be responsible for the maintenance of the response. It is induced more slowly than CuoB, and its expression levels reach a plateau. CuoC also plays an important role in development, especially in the sporulation process.
These results clearly demonstrate that along with differential induction with increased copper concentrations, differences in timing of expression to adjust the cells to the Cu-regulated adaptive response justify the redundancy of these MCOs in M. xanthus.
CuoB is cotranscribed in the same mRNA with an outer membrane efflux protein and corE, an ECF RNA polymerase sigma factor. Promoter selection by alternative forms of RNA polymerase holoenzyme containing different sigma factor subunits is a primary mechanism for regulating transcription of specific genes in response to a certain stimulus or stress conditions.
ECF ¿ factors are small regulatory proteins that are quite divergent in sequence content, and CorE has the described regions conserved in this group. At least three common characteristics are shared among many ECFs. They often recognize promoter elements with an AAC motif in the -35 region. In most cases, ECF are cotranscribed with a transmembrane anti-¿ having an extracytoplasmic sensory domain and an intracellular inhibitory domain. Finally and most importantly, ECF are mainly associated with extracellular functions, being related with regulation of periplasmic stress and heat shock, iron transport, metal ion efflux systems, alginate secretion and synthesis of membrane-localized carotenoids.
As many members of this family, CorE is involved in metal homeostasis. It is necessary for vegetative and developmental growth in the presence of external copper, and completely regulates expression of cuoB and copB (a PIB-ATPase), also involved in copper homeostasis. Genes under control of CorE exhibits a peculiar expression profile, with a rapid induction after the copper addition, which reaches a peak at 2 h of the metal supplementation. Western blot analyses have shown that CorE levels are maintained almost constantly in the cells after the copper addition for periods of times longer than 24 h, meaning that some kind of inhibition is inactivating CorE after a copper shock. Therefore, this sigma factor is going to act when cells detect this metal stress.
CorE is different from other related ECF sigma factors previously described. Positive autorregulation does not contribute significantly to its constitutive expression. However, CorE autorregulates itself expression in a copper dependent way, condition which also activates CorE-regulated genes. It doesn't have a cognate antisigma cotranscribed, and no expression of CorE regulated gene cuoB was detected when CorE was overexpressed, as it should be expected if an antisigma would be sequestering CorE. Thus, this ECF sigma factor does not work with an antisigma factor. Another interesting result is the mechanism of action: it is activated by Cu2+, copper oxidation state needed by CorE to bind DNA, and Cu+ inhibits its function. As far as we know, no sigma factor has been described to need metal for allowing the RNA polymerase to initiate transcription.
The peculiarity of this ECF sigma factor lies in an extra segment in the C-terminal region containing 6 cysteines, which has been named CRD (cysteine rich domain). Deletion of the CRD domain and point mutations in those cysteines have shown that this portion is required to promote gene expression and has allowed to identify the residues required to detect Cu2+ (abolish transcription) and Cu+ (the expression does not peak at 2 h).
