Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution.

Functional and small-angle X-ray scattering studies of a new stationary phase survival protein E (SurE) from Xylella fastidiosa--evidence of allosteric behaviour.

The genome data of bacterium Xylella fastidiosa strain 9a5c has identified several orfs related to its phytopathogenic adaptation and survival. Among these genes, the surE codifies a survival protein E (XfSurE) whose function is not so well understood, but functional assays in Escherichia coli revealed nucleotidase and exopolyphosphate activity. In the present study, we report the XfSurE protein overexpression in E. coli and its purification. The overall secondary structure was analyzed by CD. Small-angle X-ray scattering and gel filtration techniques demonstrated that the oligomeric state of the protein in solution is a tetramer. In addition, functional kinetics experiments were carried out with several monophosphate nucleoside substrates and revealed a highly positive cooperativity. An allosteric mechanism involving torsion movements in solution is proposed to explain the cooperative behaviour of XfSurE. This is the first characterization of a SurE enzyme from a phytopathogen organism and, to our knowledge, the first solution structure of a SurE protein to be described.

Molecular and cytogenetic characterization of an AT-rich satellite DNA family in Urvillea chacoensis Hunz. (Paullinieae, Sapindaceae).

Urvillea chacoensis is a climber with 2n = 22 and some terminal AT-rich heterochromatin blocks that differentiate it from other species of the genus. The AT-rich highly repeated satellite DNA was isolated from U. chacoensis by the digestion of total nuclear DNA with HindIII and XbaI and cloned in Escherichia coli. Satellite DNA structure and chromosomal distribution were investigated. DNA sequencing revealed that the repeat length of satDNA ranges between 721 and 728 bp, the percentage of AT-base pairs was about 72-73% and the studied clones showed an identity of 92.5-95.9%. Although this monomer has a tetranucleosomal size, direct imperfect repetitions of ~180 bp subdividing it in four nucleosomal subregions were observed. The results obtained with FISH indicate that this monomer usually appears distributed in the terminal regions of most chromosomes and is associated to heterochromatin blocks observed after DAPI staining. These observations are discussed in relation to the satellite DNA evolution and compared with other features observed in several plant groups.