SUFU promotes GLI activity in a Hedgehog-independent manner in pancreatic cancer.

Aberrant activation of the Hedgehog (Hh) signaling pathway, through which the GLI family of transcription factors (TF) is stimulated, is commonly observed in cancer cells. One well-established mechanism of this increased activity is through the inactivation of Suppressor of Fused (SUFU), a negative regulator of the Hh pathway. Relief from negative regulation by SUFU facilitates GLI activity and induction of target gene expression. Here, we demonstrate a novel role for SUFU as a promoter of GLI activity in pancreatic ductal adenocarcinoma (PDAC). In non-ciliated PDAC cells unresponsive to Smoothened agonism, SUFU overexpression increases GLI transcriptional activity. Conversely, knockdown (KD) of SUFU reduces the activity of GLI in PDAC cells. Through array PCR analysis of GLI target genes, we identified B-cell lymphoma 2 (BCL2) among the top candidates down-regulated by SUFU KD. We demonstrate that SUFU KD results in reduced PDAC cell viability, and overexpression of BCL2 partially rescues the effect of reduced cell viability by SUFU KD. Further analysis using as a model GLI1, a major TF activator of the GLI family in PDAC cells, shows the interaction of SUFU and GLI1 in the nucleus through previously characterized domains. Chromatin immunoprecipitation (ChIP) assay shows the binding of both SUFU and GLI1 at the promoter of BCL2 in PDAC cells. Finally, we demonstrate that SUFU promotes GLI1 activity without affecting its protein stability. Through our findings, we propose a novel role of SUFU as a positive regulator of GLI1 in PDAC, adding a new mechanism of Hh/GLI signaling pathway regulation in cancer cells.

GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass.

A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior.

Draft Genome Sequence of Cellulolytic and Xylanolytic Paenibacillus sp. A59, Isolated from Decaying Forest Soil from Patagonia, Argentina.

Paenibacillus sp. A59 was isolated from decaying forest soil in Argentina and characterized as a xylanolytic strain. We report the draft genome sequence of this isolate, with an estimated genome size of 7 Mb which harbor 6,424 coding sequences. Genes coding for hydrolytic enzymes involved in lignocellulose deconstruction were predicted.

Purification and characterization of a GH43 ß-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria.

The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40 °C and Km and Kcat values were 2.92 mM and 1.32 seg(-1), respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s(-1). These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.

Evidence for plasmid-mediated tetracycline resistance in Paenibacillus larvae, the causal agent of American Foulbrood (AFB) disease in honeybees.

Paenibacillus larvae is the causal agent of American Foulbrood (AFB) disease, the most virulent bacterial disease of honeybee (Apis mellifera L.) brood. Oxytetracycline is the main antibiotic used for prevention and control of AFB. Using the polymerase chain reaction, isolates were screened for the presence of the tetracycline resistance tet(K) and tet(L) determinants. Four isolates (5%), which correlated with the Tc-resistant phenotypes, were found to carry the tet(K) determinant, whereas none carried the tet(L) determinant. P. larvae cells were also screened for the presence of extrachromosomal DNA and evidence obtained that tetracycline resistance is plasmid-encoded. A few P. larvae isolates were found to be able to transfer the tet(K) determinant to Bacillus subtilis, suggesting that a conjugation mechanism may be involved in the transfer of the tetracycline-resistant phenotype. Minimum inhibitory concentrations to tetracycline were determined for 75 isolates of P. larvae from different geographical origins and found to range between 0.062 and 128 microg tetracyclineml(-1), with MIC(50) and MIC(90) values of 1 and 4, respectively. According to results from P. larvae populations, isolates could be considered as susceptible when their MICs were <4, intermediate for MICs values 4-8 and resistant for MICs > or = 16. To our knowledge, this is the first report of Tc(r)Paenibacillus species carrying a tet(K) gene, and also the first record of P. larvae strains carrying tet(K) determinants and its correlation with the presence of extrachromosomal DNA.

Rhizobium tropici response to acidity involves activation of glutathione synthesis.

Rhizobium tropici CIAT899 displays intrinsic tolerance to acidity, and efficiently nodulates Phaseolus vulgaris at low pH. By characterizing a gshB mutant strain, glutathione has been previously demonstrated to be essential for R. tropici tolerance to acid stress. The wild-type gshB gene region has been cloned and its transcription profile has been characterized by using quantitative real-time PCR and transcriptional gene fusions. Activation of the gshB gene under acid-stress conditions was demonstrated. gshB is also induced by UV irradiation. Upstream from gshB a putative sigma(70) promoter element and an inverted repeat sequence were identified, which are proposed to be involved in expression under neutral and acidic conditions, respectively. Gel retardation assays indicate that transcription in acid conditions may involve protein binding to an upstream regulatory region.

GuaB activity is required in Rhizobium tropici during the early stages of nodulation of determinate nodules but is dispensable for the Sinorhizobium meliloti-alfalfa symbiotic interaction.

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.

Involvement of intestinal inducible nitric oxide synthase (iNOS) in the early stages of murine salmonellosis.

Local induction of inducible nitric oxide synthase (iNOS) and apoptosis was examined in the intestine of mice infected with virulent Salmonella enterica serovar Enteritidis 5694 (S. enteritidis) and its attenuated derivative mutant E/1/3. Both, intestinal iNOS mRNA expression and iNOS activity showed a peak at 4 h only in animals receiving the virulent S. enteritidis. Aminoguanidine treatment abrogated intestinal epithelial damage produced by virulent S. enteritidis and diminished apoptosis at the tips of the villi. Unlike the virulent strain, mutant E/1/3 induced massive iNOS expression in Peyer's patches, these findings may be related to its protective capacity. Our results suggest that intestinal iNOS participates in the early response to intestinal infection and that the final effect depends on the nature of the insult.