Rodolpho M. Albano

Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5

BackgroundGluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins.ResultsGluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat.ConclusionThe genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

ABSTRACT Highly efficient production of a Thermomyces lanuginosus IOC-4145 β-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter. P. pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 23 factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in P. pastoris makes this expression system attractive for fermentor growth and industrial applications.

Environmental Shaping of Sponge Associated Archaeal Communities

Background Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood. Methodology/Principal Findings We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA) gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA) generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum. Conclusion/Significance The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition of their associated archaeal communities, thereby improving their fitness in impacted environments.

Archaeal communities in a tropical estuarine ecosystem: Guanabara Bay, Brazil.

Guanabara Bay is an eutrophic estuarine system located in a humid tropical region surrounded by the second largest metropolitan area of Brazil. This study explores the contrasting environmental chemistry and microbiological parameters that influence the archaeaplankton diversity in a pollution gradient in Guanabara Bay ecosystem. The environments sampled ranged from completely anoxic waters in a polluted inner channel to the adjacent, relatively pristine, coastal Atlantic Ocean. Partial archaeal 16S rDNA sequences in water samples were retrieved by polymerase chain reaction (PCR) and analyzed using denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. Sequences were subjected to phylogenetic and diversity analyses. Community structure of the free-living archaeal assemblages was different from that of the particle-attached archaea according to DGGE. Gene libraries revealed that phylotype identification was consistent with environmental setting. Archaeal phylotypes found in polluted anoxic waters and in more pristine waters were closely related to organisms that have previously been found in these environments. However, inner bay archaea were related to organisms found in oil, industrial wastes, and sewage, implying that water pollution controls archaea communities in this system. The detection of a substantial number of uncultured phylotypes suggests that Guanabara Bay harbors a pool of novel archaeaplankton taxa.

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium‐independent (iPLA2) and cytosolic phospholipase A2 (cPLA2), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC‐1 line infected with the ExoU‐producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA2 inhibitor, as well as significant amounts of the cyclooxygenase (COX)‐derived prostaglandins PGE2 and PGI2. Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non‐infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 104 cfu of PA103 exhibited a marked influx of inflammatory cells and PGE2 release that could be significantly reduced by indomethacin, a non‐selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid–mediated inflammatory response of host organisms.

CYP2A6 polymorphisms and risk for tobacco-related cancers.

Tobacco consumption is the main identifiable risk to cancer, contributing to the majority of tumors in upper aerodigestive tissues. The psychoactive compound responsible for tobacco addiction, nicotine and the potent carcinogens present at high concentrations either in cigarette mainstream smoke or in smokeless tobacco products, 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) and N-nitrosonornicotine (NNN) can be metabolized by CYP2A6. CYP2A6 is expressed in many aerodigestive tissues with high interindividual variability. The CYP2A6 gene is highly polymorphic and CYP2A6 alleles coding for enzymes with altered expression or metabolic capacity produce alterations in nicotine metabolism in vivo and seem to influence smoking behavior. These polymorphisms may change the rate of NNK and NNN activation and, therefore, may influence cancer risk associated with tobacco consumption. However, to date only a few and inconclusive studies have addressed the risk that a given CYP2A6 polymorphism presents for the development of tobacco-related tumors. Most, but not all, show a reduced risk associated with alleles that result in decreased enzyme activity. The overlapping substrate specificity and tissue expression between CYP2A6 and the highly similar CYP2A13 may add to the conflicting results observed. The intricate regulation of CYP2A6 and the variation of structurally different chemical compounds capable of inhibiting CYP2A enzymes also add to the complexity. Finally, the interaction between polymorphisms of genes that code for CYP2A6, CYP2A13 and other potent carcinogen-metabolizing CYP enzymes may help to determine individuals that are at higher risk of developing tumors associated with tobacco consumption.

Isolation of NDM-producing Providencia rettgeri in Brazil

Laboratório de Pesquisa em Infecção Hospitalar (LAPIH), Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro, RJ, Brazil; Departamento de Bioquı́mica, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS IPB-LACEN-RS), Porto Alegre, RS, Brazil; Hospital Nossa Senhora da Conceição, Porto Alegre, RS, Brazil

Combination of cetuximab with chemoradiation, trastuzumab or MAPK inhibitors: mechanisms of sensitisation of cervical cancer cells

Background:Cervical cancer (CC) annually kills 288 000 women worldwide. Unfortunately, responses to chemoradiation are partial and are of short duration. As anti-EGFR monoclonal antibodies sensitise tumours, we investigated cetuximabs toxicity plus chemoradiation on CC cells, which express different EGFR levels.Methods:EGFR, HER2, AKT and MAPK expression and phosphorylation status were determined by western blotting. Cytotoxicity was assessed by MTT or clonogenic assays (CA) in cell lines treated with cetuximab alone or in combinations.Results:Cetuximab with cisplatin and radiation achieved maximum cytotoxic effects for A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in CA. Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells. To check whether further EGFR, HER2 or MAPK inhibition would improve cetuximabs cytotoxicity, we combined it with an EGFR tyrosine kinase inhibitor (TKI), trastuzumab or a MEK1/2 inhibitor (PD98059). In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation. However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines.Conclusion:Our data suggest that cetuximab combined with chemoradiation, trastuzumab or MAPK inhibitors has useful applications for CC treatment, independently of EGFR expression.

Achromobacter xylosoxidans: Characterization of Strains in Brazilian Cystic Fibrosis Patients

ABSTRACT We investigated the possibility of cross-infection among cystic fibrosis patients in two Brazilian reference centers. Achromobacter xylosoxidans isolates (n = 122) were recovered over a 5-year period from 39 patients. Isolates were genetically heterogeneous, but one genotype was present in 56% of the patients, suggesting that cross-infection may have occurred.

Detection of cytotoxin genotypes of Helicobacter pylori in stomach, saliva and dental plaque

BACKGROUND The aim of this study was to detect the presence of Helicobacter pylori and its virulent cagA genes in the oral cavity of individuals with upper gastric diseases. Sixty-two individuals (42+/-2.3 years) with dispepsy symptoms, referred for gastroscopy and who were H. pylori positive in the gastric biopsy, were recruited and separated in two groups: case group-individuals with gastric disease (n = 30); control group-individuals with no gastric disease (n = 32); saliva, dental plaque and biopsy samples were collected from all individuals. Oral and biopsy samples were analyzed by PCR using specific primers for H. pylori 16S ribosomal and cagA genes. PCR products were sequenced for DNA homology confirmation. H. pylori was detected neither in dental plaque nor in saliva in the control group. In the case group H. pylori DNA was detected in 16/30 (53.3%) saliva samples and in 11/30 (36.6%) dental plaque samples. The cagA gene was detected in 13/30 (43.3%) gastric biopsies, in 7/16 (43.8%) saliva samples, and in 3/11 (27.3%) dental plaque samples. Eighteen (60.0%) individuals in the case group were H. pylori positive both in oral and biopsy samples, and 8 (26.6%) of those were positive for cagA-H. pylori DNA. H. pylori and its virulent clone showed a higher prevalence in the oral cavity of individuals in the case group than in the control group (p < 0.05). Our results suggest that dental plaque and saliva may serve as temporary reservoir for H. pylori and its virulent cagA variant in individuals with gastric disease.