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. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)

Different protocols to produce artificial dentine carious lesions in vitro and in situ: hardness and mineral content correlation

This study compared dentine demineralization induced by in vitro and in situ models, and correlated dentine surface hardness (SH), cross-sectional hardness (CSH) and mineral content by transverse microradiography (TMR). Bovine dentine specimens (n = 15/group) were demineralized in vitro with the following: MC gel (6% carboxymethylcellulose gel and 0.1 m lactic acid, pH 5.0, 14 days); buffer I (0.05 m acetic acid solution with calcium, phosphate and fluoride, pH 4.5, 7 days); buffer II (0.05 m acetic acid solution with calcium and phosphate, pH 5.0, 7 days), and TEMDP (0.05 m lactic acid with calcium, phosphate and tetraethyl methyl diphosphonate, pH 5.0, 7 days). In an in situ study, 11 volunteers wore palatal appliances containing 2 bovine dentine specimens, protected with a plastic mesh to allow biofilm development. The volunteers dripped a 20% sucrose solution on each specimen 4 times a day for 14 days. In vitro and in situ lesions were analyzed using TMR and statistically compared by ANOVA. TMR and CSH/SH were submitted to regression and correlation analysis (p < 0.05). The in situ model produced a deep lesion with a high R value, but with a thin surface layer. Regarding the in vitro models, MC gel produced only a shallow lesion, while buffers I and II as well as TEMDP induced a pronounced subsurface lesion with deep demineralization. The relationship between CSH and TMR was weak and not linear. The artificial dentine carious lesions induced by the different models differed significantly, which in turn might influence further de- and remineralization processes. Hardness analysis should not be interpreted with respect to dentine mineral loss

Genome Sequence of Exiguobacterium antarcticum B7, Isolated from a Biofilm in Ginger Lake, King George Island, Antarctica

Exiguobacterium antarcticum is a psychotropic bacterium isolated for the first time from microbial mats of Lake Fryxell in Antarctica. Many organisms of the genus Exiguobacterium are extremophiles and have properties of biotechnological interest, e. g., the capacity to adapt to cold, which make this genus a target for discovering new enzymes, such as lipases and proteases, in addition to improving our understanding of the mechanisms of adaptation and survival at low temperatures. This study presents the genome of E. antarcticum B7, isolated from a biofilm sample of Ginger Lake on King George Island, Antarctic peninsula.

Analysis of the biofilm proteome of Xylella fastidiosa

Abstract Background Xylella fastidiosa is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of X. fastidiosa strain 9a5c, in comparison to planktonic growth condition. Results We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of X. fastidiosa. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments. Conclusions We observed overexpression of proteins related to quorum sensing, proving the existence of communication between cells, and thus the development of structuring the biofilm (mature biofilm) leading to obstruction of vessels and development of disease. This paper reports a first proteomic analysis of mature biofilm of X. fastidiosa, opening new perspectives for understanding the biochemistry of mature biofilm growth in a plant pathogen.