Gammaproteobacterium Thiomicrospira crunogena thrives at deep-sea vents despite extreme oscillations in the environmental supply of dissolved inorganic carbon (DIC; =CO2 + HCO3- + CO3-2). Survival in this habitat is likely aided by the presence of a carbon concentrating mechanism (CCM). Though CCMs are well-documented in cyanobacteria, based on this study T. crunogena is the first chemolithoautotroph to have a physiologically characterized CCM. T. crunogena is capable of rapid growth in the presence of 20 micrometers DIC, has the ability to use both extracellular HCO3- and CO2, and generates intracellular DIC concentrations 100-fold greater than extracellular, all of which are consistent with a CCM analogous to those present in cyanobacteria. Interestingly, however, the T.crunogena genome lacks apparent orthologs of many of the components of the cyanobacteria CCM (e.g., HCO3- transporters). However, despite this lack, several candidate genes were identified during genome annotation as likely to play a role in DIC uptake and fixation (three carbonic anhydrase genes: alpha-CA, beta-CA, and csoSCA, as well as genes encoding three RubisCO enzymes: cbbLS, CScbbLS, and cbbM, which encode a cytoplasmic form I RubisCO, a carboxysomal form I RubisCO, and a form II RubisCO, respectively). In order to clarify their possible roles in DIC uptake and fixation, alpha-CA, beta-CA and csoSCA transcription by low-DIC and high-DIC T. crunogena were assayed by qRT PCR, heterologous expression in E. coli, and potentiometric assays of low-DIC and high-DIC T. crunogena. Transcription of alpha-CA and beta-CA were not sensitive to the DIC concentration available during growth. When overexpressed in E.coli, carbonic anhydrase activity was detectable, and it was possible to measure the effects of the classical carbonic anhydrase inhibitors ethoxyzolamide and acetazolamide, as well as dithiothreitol (DTT; recently determined to be a carboxysomal CA inhibitor). The alpha-CA was sensitive to both of the classical inhibitors, but not DTT. Beta-CA was insensitive to all inhibitors tested, and the carboxysomal carbonic anhydrase was sensitive to both ethoxyzolamide and DTT. The observation that the CA activity measureable potentiometrically with intact T. crunogena cells is sensitive to classical inhibitors, but not DTT, strongly suggests the alpha-CA is extracellular. The presence of carbonic anhydrase activity in crude extracts of high-DIC cells that was resistant to classical inhibitors suggests that beta-CA may be more active in high-DIC cells. Incubating cells with ethoxyzolamide (which permeates cells rapidly) resulted in inhibition of carbon fixation, but not DIC uptake, while incubation with acetazolamide (which does not permeate cells rapidly) had no apparent effect on either carbon fixation or DIC uptake. The observations that inhibition of alpha-CA has no effect on DIC uptake and fixation, and that the beta-CA is not transcribed more frequently under low-DIC conditions, make it unlikely that either play a role in DIC uptake and fixation in low-DIC cells. Further studies are underway to determine the roles of alpha-CA and beta-CA in T. crunogena. To assay the entire genome for genes transcribed more frequently under low-DIC conditions, and therefore likely to play a role in the T. crunogena CCM, oligonucleotide arrays were fabricated using the T. crunogena genome sequence. RNA was isolated from cultures grown in the presence of both high (50 mM) and low (0.05 mM) concentrations of DIC, directly labeled with cy5 fluorophore, and hybridized to microarrays. Genes encoding the three RubisCO enzymes present in this organism demonstrated differential patterns of transcription consistent with what had been observed previously in Hydrogenovibrio marinus. Genes encoding two conserved hypothetical proteins were also found to be transcribed more frequently under low-DIC conditions, and this transcription pattern was verified by qRT-PCR. Knockout mutants are currently being generated to determine whether either gene is necessary for growth under low-DIC conditions. Identifying CCM genes and function in autotrophs beyond cyanobacteria will serve as a window into the physiology required to flourish in microbiallydominated ecosystems where noncyanobacterial primary producers dominate.

In situ assays of N2 fixation activity, using the acetylene reduction technique, were performed in four successional stages of a Northern Wasatch Mountain subalpine forest ecosystem, elevation 2,800 m. Emphasis was made on rhizosphere fixation in association with Antennaria microphylla and Achillea millefolium. The vegetation period was approximately 100 days. Assays were performed in Saran bags. A defined amount of propane was injected at initiation of the assay and acetylene was generated from CaC2. Samples were analyzed for ethylene and propane. Data were evaluated assuming that the ethylene production was directly proportional to the increase in the ratio of ethylene to propane in the samples. Input of N by soil free-living N2 fixers in the meadow, the aspen, the fir and the spruce was 0.5, 0.3, 0.2 and 0.3 kg N ha-1 y-1, respectively. A higher activity in the presence of plant, as compared to the soil activity, was measured in 10 of the 16 assays, however, the increase was significant at three testings only. This might indicate a contribution by rhizosphere N2 fixation, but to an extent lower than was expected and therefore not detectable with the method used Leakage of gases from the test device was not corrected for in the method used for evaluation of data. This introduces and overestimation of the obtained activities that increases exponentially with a more rapid effusion rate. Correction for effusion from a closed device can be made provided quantitative analyses of the tracer gas. To determine a small difference between enclosures with and without plants the accuracies in the effusion rates must be high. Quantitative analyses were not required for evaluation according to the method used and therefore, the obtained effusion rates have too wide standard deviations for correction of the effusion rate. It was shown that the determined effusion rates with corresponding standard deviations might obscure a low rhizosphere N2 fixation activity. Acetylene reduction assays performed in open devices can not easily be corrected for diffusion of gases. The initial very rapid diffusion from an open device leads to a vastly overestimated acetylene reduction activity, when the diffusion is not corrected for.

The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.

Thesis: Ph. D., Joint Program in Physical Oceanography (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 155-162). / This work concerns the nitrogen fixation and abundance of Trichodesmium colonies in the western subtropical-tropical North Atlantic and their connections with physical processes. Data were collected in fall 2010 and spring 2011, primarily using the Video Plankton Recorder (VPR). A data processing procedure for estimating the abundance of rare taxa was devised to take advantage of the accuracy of manual classification and the effort savings of automatic classification. The procedure entails selecting a subset of the original dataset, classifying it with automated software, and then manually correcting each classification. The method was validated through comparisons with fully classified VPR data and with abundance data based on microscopic enumeration on preserved samples. Correlations of Trichodesmium colony abundance with the eddy field emerged in two subsets of the VPR observations. In fall 2010, local maxima in abundance were observed in a series of cyclones. We hypothesized Ekman transport convergence/ divergence in cyclones/anticyclones as a driving mechanism. We investigated the process using idealized three-dimensional models of buoyant colonies in eddies. Elevated abundances in anticyclones in spring 2011 were correlated with anomalously fresh water, suggesting riverine input as a driver of the relationship. Finally, we evaluated the hypothesis of Davis and McGillicuddy (2006) that Trichodesmium nitrogen fixation in the North Atlantic may be underestimated by conventional sampling methods, based on their VPR observations of higher than expected colony abundances at depth in the subtropical North Atlantic. A bio-optical model was developed based on carbon-normalized nitrogen fixation rates measured in fall 2010 and spring 2011 and used to estimate nitrogen fixation over the VPR transects. Estimates of abundance and nitrogen fixation were similar in magnitude and vertical and geographical distribution to estimates compiled in a database by Luo et al. (2012). In the mean, VPR-based estimates of volume-specific nitrogen fixation rates at depth in the tropical North Atlantic were not inconsistent with estimates derived from conventional sampling methods. Based on this analysis, if Trichodesmium nitrogen fixation is underestimated, it is unlikely that it is attributable to underestimation of deep colony abundances due to mechanical disturbance during net-based sampling. / by Elise Marie Black Olson. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18332号 / 農博第2057号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4839(農学部図書室) / 31190 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 渡邊 隆司, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19778号 / 農博第2174号 / 新制||農||1041(附属図書館) / 学位論文||H28||N4994(農学部図書室) / 32814 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Legumes are unique plants because they form special structures “nodules”, via symbiotic relationships with rhizobial bacteria present in the soil. Once rhizobia mature inside nodules, they fix atmospheric nitrogen providing a source of bioavailable nitrogen to the plant. To discover novel genetic components involved in the legume-rhizobia symbiosis by using forward genetic screening, we have isolated Medicago truncatula Tnt1 insertion mutants in the R108 ecotype, which are defective in nodule development and symbiotic nitrogen fixation in response to Sinorhizobium meliloti. Out of three mutants NF11044, NF11217 and NF8324, one of the mutants showed brown nodules and Fix- phenotype that is defective in symbiotic nitrogen fixation. The other two mutants showed white nodules and Fix- phenotype, also indicator of defects in symbiotic nitrogen fixation. To identify the underlying mutation causing the phenotype, we have developed molecular genetic markers by obtaining genomic sequences flanking the Tnt1 insertions by TAIL-PCR and Illumina sequencing. To carry out co-segregation analysis, back-crossed BC1F2 segregating populations were obtained. These are being phenotyped, genotyped and analyzed for co-segregation of the phenotype with the Tnt1 genetic markers. Back-crossing also has the effect of reducing the Tnt1 insertions, which are not linked to the nodulation defective phenotypes. Out of the three mutants, NF8324 harbors exactly the same insertion as in the rsd-1 Tnt1 mutant NF11265. The defect in NF11217 is caused by a Tnt1 insertion in the previously described PLC gene; the site of this insertion is close to that found in a different mutant, NF0217. For mutant NF11044, we developed linkage markers that place the defective locus on chromosome 7. To further characterize co-segregation in NF11044, a mapping population has been created by crossing the mutant with other ecotypes: A17 and A20. We tested mutants and wild type plants with linkage marker A20 X NF11044 BC1F2 that segregates 3:1(wild type: mutant). The recombination frequency ratio is similar as compared to back-crosses to ecotype R108. However, we did not observe mutant phenotypes in the A17 X NF11044 BC1F2 population. Future identification of the defective gene and functional characterization of it once it is identified will be carried out to better understand the mechanism of nodule organogenesis and symbiotic nitrogen fixation.

The Central Arctic Ocean is a repository of rich and diverse biota, whose major portion is one of the most important drivers of global biogeochemical cycles, including carbon cycling. In this study, the functional potential of the microbiota to fix carbon with alternative carbon fixation pathways were investigated along with their chemolithotrophic characteristics. Samples from two expeditions MOSAiC & SAS-Oden (2019-2021) resulted in metagenomic data consisting of about 1200 mOTUs (metagenomic Operational Taxonomic Units). Kofamscan based annotation explained by KEGG pathways database was analysed to explore prevalence of the alternative Carbon Fixation pathways across different taxa. From the six carbon fixation pathways, three were consolidated for their presence (rTCA, DC-HP, HP-HP). In order to explain the other metabolic processes that these organisms employ to survive, a functional annotation tool for metabolic pathways was used. that Reductive Tricarboxylic acid cycle pathway was found to be most present and observed in 5 out of 6 mOTUs selected from filtering the dataset. The taxa include bacterial phyla Proteobacteria, Actinobacteria, Chloroflexota and Marinisomatota and archaeal phyla Thermoplasmota. However, for the other pathways and less studied organisms less resolution were observed across the dataset for the presence of other pathways. These CFPs found were also supported by oxidation of inorganic compounds with high redox potentials. This study provides a glimpse of the metabolic potential of the Central Arctic Ocean microbiota, shines light on the importance of understanding and unravelling the intricacies of this rich and diverse environment.

The in vitro comparison of monotonic and cyclic mechanical properties of equine proximal interphalangeal joint arthrodeses stabilized using an open or closed technique and application of 2 abaxial transarticular lag screws combined with either an axial 4.5mm narrow 3-hole LC-DCP with 5.5mm cortical screws a 4.5mm narrow 3-hole LCP with 5.0mm locking screws. Limbs were tested for cyclic fatigue at 20,0000cycles and then in single-cycle to failure under 3-point, dorsopalmar bending. There were no significant differences in stiffness and single-cycle to failure values between the LCP and LC-DCP constructs, with or without PIPJ collateral ligament transaction. There was no interaction between the open and closed techniques, nor between plate types on force or stiffness at failure.