Biofilm formation is one of the main causes for the persistence of Acinetobacter baumannii, a pathogen associated with severe infections and outbreaks in hospitals. Here, we performed comparative proteomic analyses (2D-DIGE and MALDI-TOF/TOF and iTRAQ/SCX-LCÀMS/MS) of cells at three different conditions: exponential, late stationary phase, and biofilms. These results were compared with alterations in the proteome resulting from exposure to a biofilm inhibitory compound (salicylate). Using this multiple-approach strategy, proteomic patterns showed a unique lifestyle for A. baumannii biofilms and novel associated proteins. Several cell surface proteins (such as CarO, OmpA, OprD-like, DcaP-like, PstS, LysM, and Omp33), as well as those involved in histidine metabolism (like Urocanase), were found to be implicated in biofilm formation, this being confirmed by gene disruption. Although L-His uptake triggered biofilms efficiently in wild-type A. baumannii, no effect was observed in Urocanase and OmpA mutants, while a slight increase was observed in a CarO deficient strain. We conclude that Urocanase plays a crucial role in histidine metabolism leading to biofilm formation and that OmpA and CarO can act as channels for L-His uptake. Finally, we propose a model in which novel proteins are suggested for the first time as targets for preventing the formation of A. baumannii biofilms.

BackgroundAcinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate A. baumannii genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of A. baumannii.ResultsWe describe here a rapid and simple method of obtaining A. baumannii mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (omp33, oxyR, and soxR) by this method. In addition, we disrupted one of these genes (omp33) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining A. baumannii mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in A. baumannii.ConclusionsThis study provides a rapid and simple method of obtaining stable mutants of A. baumannii free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.

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The maintenance of genome integrity is crucial in seeds, due to the constant challenge of several endogenous and exogenous factors. The knowledge concerning DNA damage response and chromatin remodeling during seed development is still scarce, especially in Phaseolus vulgaris L. A transcriptomic profiling of the expression of genes related to DNA damage response/chromatin remodeling mechanisms was performed in P. vulgaris seeds at four distinct developmental stages, spanning from late embryogenesis to seed desiccation. Of the 14,001 expressed genes identified using massive analysis of cDNA ends, 301 belong to the DNA MapMan category. In late embryogenesis, a high expression of genes related to DNA damage sensing and repair suggests there is a tight control of DNA integrity. At the end of filling and the onset of seed dehydration, the upregulation of genes implicated in sensing of DNA double-strand breaks suggests that genome integrity is challenged. The expression of chromatin remodelers seems to imply a concomitant action of chromatin remodeling with DNA repair machinery, maintaining genome stability. The expression of genes related to nucleotide excision repair and chromatin structure is evidenced during the desiccation stage. An overview of the genes involved in DNA damage response and chromatin remodeling during P. vulgaris seed development is presented, providing insights into the mechanisms used by developing seeds to cope with DNA damage.

Background: Acinetobacter baumannii is a nosocomial pathogen that has been associated with outbreak infections in hospitals. Despite increasing awareness about this bacterium, its proteome remains poorly characterised, however recently the complete genome of A. baumannii reference strain ATCC 17978 has been sequenced. Here, we have used 2-DE and MALDI-TOF/TOF approach to characterise the proteome of this strain.

Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.

The knowledge on post-transcriptional regulation mechanisms implicated in seed development (SD) is still limited, particularly in one of the most consumed grain legumes, Phaseolus vulgaris L. We explore for the first time the miRNA expression dynamics in P. vulgaris developing seeds. Seventy-two known and 39 new miRNAs were found expressed in P. vulgaris developing seeds. Most of the miRNAs identified were more abundant at 10 and 40 days after anthesis, suggesting that late embryogenesis/early filling and desiccation were SD stages in which miRNA action is more pronounced. Degradome analysis and target prediction identified targets for 77 expressed miRNAs. While several known miRNAs were predicted to target HD-ZIP, ARF, SPL, and NF-Y transcription factors families, most of the predicted targets for new miRNAs encode for functional proteins. MiRNAs-targets expression profiles evidenced that these miRNAs could tune distinct seed developmental stages. MiRNAs more accumulated at early SD stages were implicated in regulating the end of embryogenesis, postponing the seed maturation program, storage compound synthesis and allocation. MiRNAs more accumulated at late SD stages could be implicated in seed quiescence, desiccation tolerance, and longevity with still uncovered roles in germination. The miRNAs herein described represent novel P. vulgaris resources with potential application in future biotechnological approaches to modulate the expression of genes implicated in legume seed traits with impact in horticultural production systems.