Ms. var. oct. 8 No. 3b - Über die Duchoborzen, eine Religions-Secte im südlichen Rußland

Die Foliierung geht nur bis Bl. 66 ; zusätzlich gibt es die Blätter 36a-d, 39a-c, 41a-c und 44a-b.

Ms. var. oct. 8 No. 3c - Über die Duchoborzen. Erstes Heft

Die im Katalog angegebene Blattzahl weicht von der vorhandenen ab.

Mus Hs 678 - Var: Cor, et Fagott ...

par C. Oestreich

Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Functional Characterization of AtxA, the Bacillus anthracis Virulence Regulator

Coordinated expression of virulence genes in Bacillus anthracis occurs via a multi-faceted signal transduction pathway that is dependent upon the AtxA protein. Intricate control of atxA gene transcription and AtxA protein function have become apparent from studies of AtxA-induced synthesis of the anthrax toxin proteins and the poly-D-glutamic acid capsule, two factors with important roles in B. anthracis pathogenesis. The amino-terminal region of the AtxA protein contains winged-helix (WH) and helix-turn-helix (HTH) motifs, structural features associated with DNA-binding. Using filter binding assays, I determined that AtxA interacted non-specifically at a low nanomolar affinity with a target promoter (Plef) and AtxA-independent promoters. AtxA also contains motifs associated with phosphoenolpyruvate: sugar phosphotransferase system (PTS) regulation. These PTS-regulated domains, PRD1 and PRD2, are within the central amino acid sequence. Specific histidines in the PRDs serve as sites of phosphorylation (H199 and H379). Phosphorylation of H199 increases AtxA activity; whereas, H379 phosphorylation decreases AtxA function. For my dissertation, I hypothesized that AtxA binds target promoters to activate transcription and that DNA-binding activity is regulated via structural changes within the PRDs and a carboxy-terminal EIIB-like motif that are induced by phosphorylation and ligand binding. I determined that AtxA has one large protease-inaccessible domain containing the PRDs and the carboxy-terminal end of the protein. These results suggest that AtxA has a domain that is distinct from the putative DNA-binding region of the protein. My data indicate that AtxA activity is associated with AtxA multimerization. Oligomeric AtxA was detected when co-affinity purification, non-denaturing gel electrophoresis, and bis(maleimido)hexane (BMH) cross-linking techniques were employed. I exploited the specificity of BMH for cysteine residues to show that AtxA was cross-linked at C402, implicating the carboxy-terminal EIIB-like region in protein-protein interactions. In addition, higher amounts of the cross-linked dimeric form of AtxA were observed when cells were cultured in conditions that promote toxin gene expression. Based on the results, I propose that AtxA multimerization requires the EIIB-like motif and multimerization of AtxA positively impacts function. I investigated the role of the PTS in the function of AtxA and the impact of phosphomimetic residues on AtxA multimerization. B. anthracis Enzyme I (EI) and HPr did not facilitate phosphorylation of AtxA in vitro. Moreover, markerless deletion of ptsHI in B. anthracis did not perturb AtxA function. Taken together, these results suggest that proteins other than the PTS phosphorylate AtxA. Point mutations mimicking phosphohistidine (H to D) and non-phosphorylated histidine (H to A) were tested for an impact on AtxA activity and multimerization. AtxA H199D, AtxA H199A, and AtxA H379A displayed multimerization phenotypes similar to that of the native protein, whereas AtxA H379D was not susceptible to BMH cross-linking or co-affinity purification with AtxA-His. These data suggest that phosphorylation of H379 may decrease AtxA activity by preventing AtxA multimerization. Overall, my data support the following model of AtxA function. AtxA binds to target gene promoters in an oligomeric state. AtxA activity is increased in response to the host-related signal bicarbonate/CO2 because this signal enhances AtxA multimerization. In contrast, AtxA activity is decreased by phosphorylation at H379 because multimerization is inhibited. Future studies will address the interplay between bicarbonate/CO2 signaling and phosphorylation on AtxA function.

Control of the master virulence regulatory gene atxA in Bacillus anthracis

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact atxA transcript levels, and the timing and steady state level of atxA expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the atxA promoter. The AbrB-binding site has been described, but additional cis-acting control sequences have not been defined. Using transcriptional lacZ fusions, electrophoretic mobility shift assays, and Western blot analysis, the cis-acting elements and trans-acting factors involved in regulation of atxA in B. anthracis strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a trans-acting protein(s) other than AbrB negatively impacts atxA transcription when it binds specifically to a 9-bp palindrome within atxA promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the trans-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner. The identity of the trans-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the trans-acting repressor protein(s) indirectly controls B. anthracis development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both atxA- and pXO2-61-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper B. anthracis development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on atxA expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of atxA. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an abrB-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an abrB-null mutant. In addition, the abrB-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the atxA promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the abrB-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an abrB-null mutant. Overall, this work highlights the complex regulatory network that governs expression of atxA and provides an additional role for AtxA in B. anthracis development.

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

ATXA -dependent gene expression in Bacillus anthracis

The Bacillus anthracis toxin genes, cya, lef , and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. I determined that several phenotypes are associated with the atxA gene. In addition to being toxin-deficient, an atxA -null mutant grows poorly on minimal media and sporulates early compared to the parent strain. Furthermore, an atxA-null mutant has an altered 2-D gel protein profile. I used a genetic approach to find additional atxA-regulated genes. Random transcriptional lacZ fusions were generated in B. anthracis using transposon Tn 917-LTV3. Transposon-insertion libraries were screened for mutants expressing increased β-galactosidase activity in 5% CO2. Introduction of an atxA-null mutation in these mutants revealed that 79% of the CO2-regulated fusions were also atxA-dependent. DNA sequence analysis of transposon insertion sites in mutants carrying CO 2/atxA-regulated fusions revealed ten mutants harboring transposon insertions in loci distinct from the toxin genes. The majority of the tcr (toxin co-regulated) loci mapped within the pXO1 pathogenicity island. These results indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins. ^ Characterization of one tcr locus revealed a new regulatory gene, pagR. The pagR gene (300 nt) is located downstream of pag. pagR is cotranscribed with pag and is responsible for autogenous control of the operon. pagR also represses expression of cya and lef. Repression of toxin gene expression by pagR may be mediated by atxA. The steady state level of atxA mRNA is increased in a pagR mutant. Recombinant PagR protein purified from Escherichia coli did not specifically bind the promoter regions of pagA or atxA. An unidentified factor in B. anthracis crude extracts, however, was able to bind the atxA promoter in the absence of PagR or AtxA. These investigations increase our knowledge of virulence regulation in B. anthracis and ultimately will lead to a better understanding of anthrax disease. ^

Conservación de zapallos (Cucurbita pepo L.) var. Frontera : cultivo orgánico y tradicional

Objetivos: Determinar el período de conservación de zapallo orgánico respecto del tradicional, en dos condiciones de almacenamiento. Materiales y métodos: Zapallos variedad Frontera se limpiaron en seco y se pesaron individualmente para controlar pérdida de peso; se consignaron características externas. Para determinar deshidratación se midió humedad en la pulpa (estufa 105 °C). Para conservación, acondicionamiento en cajones de madera con viruta, en galpón (temperatura y humedad ambiente) y antecámara (15- 20 °C y 70-85 % HR). Duración 187 días. Comparación sensorial, al inicio y al final de la conservación, considerando: color, olor, sabor, retrogusto, jugosidad, textura y calidad general. Se utilizó planilla con escala mixta estructurada de 9 puntos y participaron jueces semientrenados (n = 10). Evaluación de datos por análisis de varianza = 0.05. Conclusiones: En las condiciones del ensayo, el período de conservación no debe ser superior a los 105 días por aparición de podredumbre. Por deshidratación superior al 60 %, debe ser inferior a 68 días para antecámara, 105 días para orgánicos de galpón y 105-126 días para tradicionales de galpón. Las características sensoriales iniciales de los zapallos orgánicos fueron mejores; al final se destacaron los tradicionales en galpón, luego los orgánicos en galpón y los conservados en antecámara presentaron características inferiores.

Caracterización del crecimiento de plantines de Brassica rapa var. pekinensis en contenedores.

Brassica rapa var. pekinensis, hakusay, es una hortaliza cuya producción puede iniciarse a partir de transplantes o siembra directa. El objetivo de este trabajo fue caracterizar el crecimiento de plantines de esta hortaliza en contenedores, previo al transplante, a través de variables morfogenéticas y de crecimiento. La biomasa total (PST), el peso seco aéreo (PSA) y el peso seco de raíces (PSR) mostraron una tendencia creciente, particular en cada caso. El área foliar (AF) se incrementó hasta estabilizarse días antes del transplante, momento en el cual se observó el efecto limitante del contenedor. Las variables morfogenéticas: razón de área foliar (RAF) y área foliar específica (AFE) descendieron hasta alcanzar valores casi constantes la semana previa al transplante. Las variables de crecimiento: índice de crecimiento relativo de la planta (ICRP) e índice de crecimiento relativo foliar (ICRF) descendieron, el primero con valores superiores hasta el día 31, hecho que se refleja a través de Gamma (Gf); la tasa absoluta de crecimiento (TAC) se incrementó hasta el día 31 del período y después cayó abruptamente; la tasa de asimilación neta (TAN) creció hasta el día 31 posterior a la siembra para luego caer al final del período. El productor podría decidir el momento del transplante como aquel definido por la detención de la expansión foliar.

Las yemas, principal reservorio de levaduras entre los órganos aéreos vegetativos y reproductivos de la vid (Vitis vinifera var. Malbec).

El objetivo de esta investigación fue suministrar nueva evidencia acerca del modelo de permanencia de las levaduras en el ciclo natural de la vid. Se efectuó la observación, la medición del número de levaduras y la descripción morfológica de los diferentes órganos aéreos de la vid. Se procedió a la recolección aséptica de muestras a campo, en yema en actividad, yema en reposo, hoja joven, hoja adulta, ritidomis, zarcillo, capullo floral, flor y fruto. Los resultados revelaron dos momentos de máxima población de levaduras: en yema cerrada a fines de otoño y en yema terminal abierta a mediados de verano. La evolución de las levaduras en función de la superficie del fruto mostró poca relación entre ambas variables, por lo que el valor a considerar sería la cantidad de levaduras por baya como unidad. La ritidomis exhibió valores muy uniformes a lo largo del ciclo vegetativo, asumiendo desde esta perspectiva el papel de reservorio de moderada importancia.

Variability in the indument of Phaseolus vulgaris var. aborigineus (Fabaceae)

The indument of Phaseolus vulgaris L. var. aborigineus (Burkart) Baudet, a native bean from Argentina, potential source for improvement of cultivated varieties, was compared between different genetic lines. Seeds collected from wild, weedy forms and presumptive hybrid individuals in two distant latitudes were sown in two sites of Buenos Aires University Campus (field and greenhouse). Two type of trichomes were found, which differenciate in length and shape. The pubescence density was significantly different between wild specimens from different latitudes and between culture sites for plants of a same procedence. This character could be involved in physical defences of these plants.

Sustrato y dosis de fertirriego en la aclimatización de vitroplantas de Agave americana var. oaxacencis

Con la finalidad de asegurar la adaptación en invernadero de plantas de Agave americana var. oaxacensis obtenidas in vitro, se evaluó el efecto de sustratos y dosis de fertirriego en la aclimatización y crecimiento de 180 plantas. Este trabajo se realizó en un invernadero del Instituto Tecnológico del Valle de Oaxaca, México, a principios de 2012. Se utilizaron plantas recién salidas del laboratorio. El experimento se estableció según el diseño completamente al azar con arreglo factorial 2 (sustratos: arena y perlita) x 5 (fertilización: 1, 25, 50, 75 y 100%). La unidad experimental fue una planta en un recipiente de plástico de 150 cm3 con 18 repeticiones. La fertilización se basó en la solución universal de Steiner y diariamente cada planta recibió a nivel de sustrato 10 mL durante 12 semanas. Se evaluó el crecimiento en hojas, tallo y raíz, realizando análisis de varianza y prueba de medias (Tukey, α = 0,05). Todas las plantas se adaptaron, pero aquellas plantas establecidas en perlita lograron los mejores crecimientos en área foliar, volumen de raíz, peso fresco total, materia seca de raíz y total de planta. El tamaño de las plantas al término de su aclimatación estuvo en relación con la cantidad de nutrimentos de la solución nutritiva.

Ensayos de rendimiento en morfina de amapola (Papaver somniferum, var. nigrum)

Fil: Wainstein, Pedro. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias