The Regulatory Network Controlling Ethanol-Induced Expression of Alcohol Dehydrogenase in the Endophyte Azoarcus sp. Strain BH72.

The habitat of the nitrogen-fixing endophyte Azoarcus sp. strain BH72 is grass roots grown under waterlogged conditions that produce, under these conditions, ethanol. Strain BH72 is well equipped to metabolize ethanol, with eight alcohol dehydrogenases (ADHs), of which ExaA2 and ExaA3 are the most relevant ones. exaA2 and exaA3 cluster and are surrounded by genes encoding two-component regulatory systems (TCSs) termed ExaS-ExaR and ElmS-GacA. Functional genomic analyses revealed that i) expression of the corresponding genes was induced by ethanol, ii) the genes were also expressed in the rhizoplane or even inside of rice roots, iii) both TCSs were indispensable for growth on ethanol, and iv) they were important for competitiveness during rice root colonization. Both TCSs form a hierarchically organized ethanol-responsive signal transduction cascade with ExaS-ExaR as the highest level, essential for effective expression of the ethanol oxidation system based on ExaA2. Transcript and expression levels of exaA3 increased in tcs deletion mutants, suggesting no direct influence of both TCSs on its ethanol-induced expression. In conclusion, this underscores the importance of ethanol for the endophytic lifestyle of Azoarcus sp. strain BH72 and indicates a tight regulation of the ethanol oxidation system during root colonization.

Flagella mediate endophytic competence rather than act as MAMPS in rice-Azoarcus sp. strain BH72 interactions.

Azoarcus sp. strain BH72 is an endophytic betaproteobacterium able to colonize rice roots without induction of visible disease symptoms. BH72 possesses one polar flagellum. The genome harbors three copies of putative fliC genes, generally encoding the major structural protein flagellin. It is not clear whether, in endophytic interactions, flagella mediate endophytic competence or act as MAMPs (microbe-associated molecular patterns) inducing plant defense responses. Therefore, possible functions of the three FliC proteins were investigated. Only fliC3 was found to be highly expressed in pure culture and in association with rice roots and to be required for bacterial motility, suggesting that it encodes the major flagellin. Endophytic colonization of rice roots was significantly reduced in the in-frame deletion mutant, while the establishment of microcolonies on the root surface was not affected. Moreover, an elicitation of defense responses related to FliC3 was not observed. In conclusion, our data support the hypothesis that FliC3 does not play a major role as a MAMP but is required for endophytic colonization in the Azoarcus-rice interaction, most likely for spreading inside the plant.

Exploring the function of alcohol dehydrogenases during the endophytic life of Azoarcus Sp. strain BH72.

The endophytic bacterium Azoarcus sp. strain BH72 is capable of colonizing the interior of rice roots, where it finds suitable physicochemical properties for multiplying and fixing nitrogen. Because these properties are poorly understood, a microtiter-plate-based screening of a transcriptional gfp (green fluorescent protein) fusion library of Azoarcus sp. grown under different conditions was performed. Monitoring of the GFP activity allowed the identification of a gene highly expressed in medium supplemented with ethanol. Sequence analysis revealed that this gene encodes a pyrrolo-quinoline quinone-dependent alcohol dehydrogenase (ADH). Inspection of the complete genome sequence of the Azoarcus sp. strain BH72 identified seven additional genes encoding putative ADH, indicating that BH72 is well equipped to survive in different environmental conditions offering various alcohols as carbon source. Analyses of these eight putative ADH showed that expression of three was induced by ethanol, of which two were also expressed inside rice roots. The fact that waterlogged plants such as rice accumulate ethanol suggests that ethanol occurs in sufficiently high concentration within the root to induce expression of bacterial ADH. Disruption of these two ADH evoked a reduced competitiveness to the wild type in colonizing rice roots internally. Thus, it is likely that ethanol is an important carbon source for the endophytic life of Azoarcus sp.

Caudal regression in adrenocortical dysplasia (acd) mice is caused by telomere dysfunction with subsequent p53-dependent apoptosis.

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutation that exhibits a pleiotropic phenotype with perinatal lethality. Mutant acd embryos have caudal truncation, vertebral segmentation defects, hydronephrosis, and limb hypoplasia, resembling humans with Caudal Regression syndrome. Acd encodes Tpp1, a component of the shelterin complex that maintains telomere integrity, and consequently acd mutant mice have telomere dysfunction and genomic instability. While the association between genomic instability and cancer is well documented, the association between genomic instability and birth defects is unexplored. To determine the relationship between telomere dysfunction and embryonic malformations, we investigated mechanisms leading to the caudal dysgenesis phenotype of acd mutant embryos. We report that the caudal truncation is caused primarily by apoptosis, not altered cell proliferation. We show that the apoptosis and consequent skeletal malformations in acd mutants are dependent upon the p53 pathway by genetic rescue of the limb hypoplasia and vertebral anomalies with p53 null mice. Furthermore, rescue of the acd phenotype by p53 deficiency is a dosage-sensitive process, as acd/acd, p53(-/-) double mutants exhibit preaxial polydactyly. These findings demonstrate that caudal dysgenesis in acd embryos is secondary to p53-dependent apoptosis. Importantly, this study reinforces a significant link between genomic instability and birth defects.

Effect of cold storage and packaging material on the major aroma components of sweet cream butter.

The major aroma compounds of commercial sweet cream AA butter quarters were analyzed by GC-olfactometry and GC-MS combined with dynamic headspace analysis (DHA) and solvent-assisted flavor evaporation (SAFE). In addition, the effect of long-term storage (0, 6, and 12 months) and type of wrapping material (wax parchment paper vs foil) on the aroma components and sensory properties of these butters kept under refrigerated (4 degrees C) and frozen (-20 degrees C) storage was evaluated. The most intense compounds in the aroma of pasteurized AA butter were butanoic acid, delta-octalactone, delta-decalactone, 1-octen-3-one, 2-acetyl-1-pyrroline, dimethyl trisulfide, and diacetyl. The intensities of lipid oxidation volatiles and methyl ketones increased as a function of storage time. Refrigerated storage caused greater flavor deterioration compared with frozen storage. The intensity and relative abundance of styrene increased as a function of time of storage at refrigeration temperature. Butter kept frozen for 12 months exhibited lower styrene levels and a flavor profile more similar to that of fresh butter compared to butter refrigerated for 12 months. Foil wrapping material performed better than wax parchment paper in preventing styrene migration into butter and in minimizing the formation of lipid oxidation and hydroxyl acid products that contribute to the loss of fresh butter flavor.

Novel polymorphisms and lack of mutations in the ACD gene in patients with ACTH resistance syndromes.

OBJECTIVE: ACTH resistance is a feature of several human syndromes with known genetic causes, including familial glucocorticoid deficiency (types 1 and 2) and triple A syndrome. However, many patients with ACTH resistance lack an identifiable genetic aetiology. The human homolog of the Acd gene, mutated in a mouse model of adrenal insufficiency, was sequenced in 25 patients with a clinical diagnosis of familial glucocorticoid deficiency or triple A syndrome. DESIGN: A 3.4 kilobase genomic fragment containing the entire ACD gene was analysed for mutations in all 25 patients. SETTING: Samples were obtained by three investigators from different institutions. PATIENTS: The primary cohort consisted of 25 unrelated patients, primarily of European or Middle Eastern descent, with a clinical diagnosis of either familial glucocorticoid deficiency (FGD) or triple A syndrome. Patients lacked mutations in other genes known to cause ACTH resistance, including AAAS for patients diagnosed with triple A syndrome and MC2R and MRAP for patients diagnosed with familial glucocorticoid deficiency. Thirty-five additional patients with adrenal disease phenotypes were added to form an expanded cohort of 60 patients. MEASUREMENTS: Identification of DNA sequence changes in the ACD gene in the primary cohort and analysis of putative ACD haplotypes in the expanded cohort. RESULTS: No disease-causing mutations were found, but several novel single nucleotide polymorphisms (SNPs) and two putative haplotypes were identified. The overall frequency of SNPs in ACD is low compared to other gene families. CONCLUSIONS: No mutations were identified in ACD in this collection of patients with ACTH resistance phenotypes. However, the newly identified SNPs in ACD should be more closely examined for possible links to disease.

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72.

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.

Effects of bone protein extract on human mesenchymal stem cells proliferation and differentiation.

Since its osteoinductive capacity has been established, demineralized bone matrix is considered a suitable alternative to bone autograft in the healing of osseous defects. The mechanisms of bone formation induction are still not fully understood. In this study we assessed the effects of a dispersion of bovine bone extract COLLOSS (BPE) with regard to proliferation and differentiation of a human mesenchymal stem cell line overexpressing human telomerase reverse transcriptase (hMSC-TERT). Proliferation rate was determined by (3)H-thymidine incorporation. The differentiation of hMSC-TERT cells to osteoblastic cells was assessed by means of measuring alkaline phosphatase activity and collagen synthesis in vitro. Both undifferentiated and osteoblast-differentiated hMSC-TERT cells were investigated for response to BPE. The metabolic responses to BPE were compared to unstimulated cells and cells stimulated with bovine collagen (COL). Undifferentiated hMSC-TERT cells responded to BPE with increased proliferation and decreased alkaline phosphatase activity. Osteoblastic differentiated hMSC-TERT cells had a diminished proliferative response and an increased alkaline phosphatase activity and collagen synthesis. Our study demonstrated significant metabolic effects of BPE on hMSC-TERT cells, which were highly dependant on the differentiated state of the cells.

Identification of genistein-inducible and type III-secreted proteins of Bradyrhizobium japonicum.

Flagellin is the bulk protein secreted by Bradyrhizobium japonicum. For easier identification of minor protein fractions, the flagellin genes bll6865 and bll6866 were deleted. Extracellular proteins of the corresponding mutant were purified and separated by 2D gel electrophoresis. Several of the protein spots were detectable only after addition of genistein to the growth medium-genistein is an isoflavone secreted by soybean that activates the expression of genes encoding a type III secretion system. These secreted proteins were not present in supernatants of mutants in which conserved genes of the type III secretion system or the regulatory gene ttsI, which is essential for activation of the type III secretion system, are deleted. Out of 22 genistein-inducible protein spots 8 different proteins could be identified by mass spectrometry. One of the proteins, Blr1752, has similarity to NopP of Rhizobium sp. strain NGR234 that is known to be secreted. Another protein is Blr1656 (GunA2) that was shown previously to have endoglucanase activity. Three proteins have similarity to subunits of the flagellar apparatus. Some proteins appeared in several separate spots indicating posttranslational modification. A conserved tts box motif was found in the putative promoter region of six genes encoding secreted proteins.

IMAGe association and congenital adrenal hypoplasia: no disease-causing mutations found in the ACD gene.

The spontaneous mouse mutant adrenocortical dysplasia (acd) is characterized by defects in the adrenals, kidneys, and gonads of adult mutant mice and by caudal dysgenesis and vertebral segmentation defects in acd embryos. This association of defects mirrors those identified in patients with known or suspected abnormalities in adrenocortical development, including adrenal hypoplasia congenita and IMAGe association. The identification of the Acd gene in mice has prompted the study of its human homolog ACD, which has recently been shown to be a regulator of telomere length. Sequencing of ACD in 15 patients revealed no coding mutations, but three novel SNPs were identified.

Mutational and transcriptional analysis of the type III secretion system of Bradyrhizobium japonicum.

Sequencing the symbiotic region of Bradyrhizobium japonicum revealed a gene cluster (tts) encoding a type III secretion system (TTSS) that is similar to those found in Mesorhizobium loti MAFF303099 and Rhizobium strain NGR234. In addition to genes that are likely to encode structural core components of the TTSS, the cluster contains several open reading frames that are found exclusively in rhizobia or that are specific to B. japonicum. Depending on the host, mutations within this cluster affected nodulation capacity to different extents. One of the genes likely encodes a transcriptional activator (TtsI) of the two-component regulatory family. Upstream of ttsI, a nod box promoter was identified. Expression of ttsI could be induced by genistein. This induction depended on the transcriptional activator protein NodW as well as the nodD1nodD2nolA gene region. TtsI was found to be involved in transcriptional regulation of the tts gene cluster. Sequence comparison revealed a conserved tts box element within putative promoter regions of several genes. Here, we propose a model of the regulatory cascade leading to the induction of the tts gene cluster.