Genome mining revealed polyhydroxybutyrate biosynthesis by Ramlibacter agri sp. nov., isolated from agriculture soil in Korea

A white-colony-forming, facultative anaerobic, motile and Gram-stain-negative bacterium, designated G-1-2-2 ^T was isolated from soil of agriculture field near Kyonggi University, Republic of Korea. Strain G-1-2-2 ^T synthesized the polyhydroxybutyrate and could grow at 10–35 °C. The phylogenetic analysis based on 16S rRNA gene sequence showed that, strain G-1-2-2 ^T formed a lineage within the family Comamonadaceae and clustered as a member of the genus Ramlibacter. The 16S rRNA gene sequence of strain G-1-2-2 ^T showed high sequence similarities with Ramlibacter ginsenosidimutans BXN5-27 ^T (97.9%), Ramlibacter monticola G-3-2 ^T (97.9%) and Ramlibacter alkalitolerans CJ661^T (97.5%). The sole respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and an unidentified phospholipid. The principal cellular fatty acids were C_16:0, cyclo-C_17:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c) and summed feature 8 (C_18:1ω7c and/or C_18:1ω6c). The genome of strain G-1-2-2 ^T was 7,200,642 bp long with 13 contigs, 6,647 protein-coding genes, and DNA G?+?C content of 68.9%. The average nucleotide identity and in silico DNA–DNA hybridization values between strain G-1-2-2 ^T and close members were?≤?81.2 and 24.1%, respectively. The genome of strain G-1-2-2 ^T showed eight putative biosynthetic gene clusters responsible for various secondary metabolites. Genome mining revealed the presence of atoB, atoB2, phaS, phbB, phbC, and bhbD genes in the genome which are responsible for polyhydroxybutyrate biosynthesis. Based on these data, strain G-1-2-2 ^T represents a novel species in the genus Ramlibacter, for which the name Ramlibacter agri sp. nov. is proposed. The type strain is G-1-2-2 ^T (=?KACC 21616 ^T?=?NBRC 114389 ^T).

     

Family organization and the wage labor transition in a Tamang community of Nepal

This paper explores familial contexts of transition to a wage labor economy using ethnographic and survey data from Tamang communities at the northern edge of Nepal's Kathmandu Valley. Historically agro-pastoralist, the Tamang of this area have experienced social watersheds drawing them into ever closer relationships with Kathmandu. The earliest was their nineteenth century induction into corvée labor for national elites; more recent has been the accelerating monetization of the twentieth century. This analysis demonstrates trends and frames hypotheses about the social structuring of this latest process, testing them at the individual level with combined ethnographic and survey data from 1028 respondents. Multivariate analyses explore the effects of birth cohort, education, domestic group status, and settlement location on participation in non-family organized wage work. Substantive findings are related to the broader historical literature on household and family with special attention to varieties of subsistence to monetized transition.     

Studies on Thermoelectric Properties of n‐type Polycrystalline SnSe1‐xSx by Iodine Doping

Iodine‐doped n‐type SnSe polycrystalline by melting and hot pressing is prepared. The prepared material is anisotropic with a peak ZT of ≈0.8 at about 773 K measured along the hot pressing direction. This is the first report on thermoelectric properties of n‐type Sn chalcogenide alloys. With increasing content of iodine, the carrier concentration changed from 2.3 × 10¹⁷ cm^?3 (p‐type) to 5.0 × 10¹⁵ cm^?3 (n‐type) then to 2.0 × 10¹⁷ cm^?3 (n‐type). The decent ZT is mainly attributed to the intrinsically low thermal conductivity due to the high anharmonicity of the chemical bonds like those in p‐type SnSe. By alloying with 10 at% SnS, even lower thermal conductivity and an enhanced Seebeck coefficient were achieved, leading to an increased ZT of ≈1.0 at about 773 K measured also along the hot pressing direction.     

Vacuolar H(+)-ATPase is expressed in response to gibberellin during tomato seed germination

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA(4+7). Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V(0) membrane sector of vacuolar H(+)-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V(1) sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.     

Relationship of Endo-[beta]-D-Mannanase Activity and Cell Wall Hydrolysis in Tomato Endosperm to Germination Rates

The endosperm tissue enclosing the radicle tip (endosperm cap) governs radicle emergence in tomato (Lycopersicon esculentum Mill.) seeds. Weakening of the endosperm cap has been attributed to hydrolysis of its mannan-rich cell walls by endo-[beta]-D-mannanase. To test this hypothesis, we measured mannanase activity in tomato endosperm caps from seeds allowed to imbibe under conditions of varying germination rates. Over a range of suboptimal temperatures, mannanase activity prior to radicle emergence increased in accordance with accumulated thermal time. Reduced water potential delayed or prevented radicle emergence but enhanced mannanase activity in the endosperm caps. Abscisic acid did not prevent the initial increase in mannanase activity, although radicle emergence was markedly delayed. Sugar composition and percent mannose (Man) content of endosperm cap cell walls did not change prior to radicle emergence under any condition. Man, glucose, and other sugars were released into the incubation solution by endosperm caps isolated from intact seeds during imbibition. Pregerminative release of Man was suppressed and the release of glucose was enhanced when seeds were incubated in osmoticum or abscisic acid; the opposite occurred in the presence of gibberellin. Thus, whereas sugar release patterns were sensitive to environmental and hormonal factors affecting germination, neither assayable endo-[beta]-D-mannanase activity nor changes in cell wall sugar composition of endosperm caps correlated well with tomato seed germination rates under all conditions.     

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.     

Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars

Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l’Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l’Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the ‘dead’ virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants.     

Two Tomato Expansin Genes Show Divergent Expression and Localization in Embryos during Seed Development and Germination

Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth.     

Mitochondrial Alternative Oxidase Maintains Respiration and Preserves Photosynthetic Capacity during Moderate Drought in Nicotiana tabacum

The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum ‘Petit Havana SR1’ wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought-stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO₂ and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity.The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons in the ubiquinone pool partition between the cytochrome (cyt) pathway (consisting of Complex III, cyt c, and Complex IV) and alternative oxidase (AOX; Finnegan et al., 2004; Millar et al., 2011; Vanlerberghe, 2013). AOX directly couples ubiquinol oxidation with O₂ reduction to water. This reduces the energy yield of respiration because, unlike Complexes III and IV, AOX is not proton pumping. Hence, AOX is an electron sink, the capacity of which is little encumbered by rates of ATP turnover. In this way, AOX might be well suited to prevent cellular over-reduction. Supporting this, transgenic Nicotiana tabacum leaves with suppressed amounts of AOX have increased concentrations of mitochondrial-localized superoxide radical (O₂−) and nitric oxide, the products that can arise when an over-reduced ETC results in electron leakage to O₂ or nitrite (Cvetkovska and Vanlerberghe, 2012, 2013).In angiosperms, AOX is encoded by a small gene family (Considine et al., 2002). In Arabidopsis (Arabidopsis thaliana), mutation or knockdown of the stress-responsive AOX1a gene family member dramatically reduces AOX protein and the capacity of the AOX respiration pathway to consume O₂. Several studies have shown that this loss of AOX capacity in Arabidopsis aox1a plants affected processes such as growth, carbon and energy metabolism, and/or the cellular network of reactive oxygen species (ROS) scavengers (Fiorani et al., 2005; Umbach et al., 2005; Watanabe et al., 2008; Giraud et al., 2008; Skirycz et al., 2010). However, in studies in which respiration was measured, it was consistently reported that the lack of AOX capacity had no significant impact on the respiration rate in the dark (R_D; Umbach et al., 2005; Giraud et al., 2008; Strodtkötter et al., 2009; Florez-Sarasa et al., 2011; Yoshida et al., 2011b; Gandin et al., 2012). The exceptions are two reports that R_D was actually higher in aox1a than in the wild type under some conditions (Watanabe et al., 2008; Vishwakarma et al., 2014). To our knowledge, how the lack of AOX affects respiration rate in the light (R_L) is not reported in Arabidopsis or other species.Numerous studies have established the importance of mitochondrial metabolism in the light to optimize photosynthesis (Hoefnagel et al., 1998; Raghavendra and Padmasree, 2003). In recent years, the potential importance of specifically AOX respiration during photosynthesis has been examined using the Arabidopsis aox1a plants (Giraud et al., 2008; Strodtkötter et al., 2009; Zhang et al., 2010; Florez-Sarasa et al., 2011; Yoshida et al., 2011a, 2011b). In general, these studies reported small perturbations of photosynthesis in standard-grown aox1a plants, including slightly lower rates of CO₂ uptake or O₂ release (Gandin et al., 2012; Vishwakarma et al., 2014), slightly higher rates of cyclic electron transport (CET; Yoshida et al., 2011b), and slightly increased susceptibility to photoinhibition after a high light treatment (Florez-Sarasa et al., 2011). Generally, these studies concluded that aox1a plants exhibit a biochemical limitation of photosynthesis, in line with the hypothesis that AOX serves as a sink for excess photogenerated reducing power, with the reductant likely reaching the mitochondrion via the malate valve (Noguchi and Yoshida, 2008; Taniguchi and Miyake, 2012). Similar to these Arabidopsis studies, we recently reported that well-watered N. tabacum AOX knockdowns grown at moderate irradiance display a slight reduced rate of photosynthesis (approximately 10%–15%) when measured at high irradiance. However, we established that the lower photosynthetic rate was the result of a stomatal rather than biochemical limitation of photosynthesis, and provided evidence that this stomatal limitation resulted from disrupted nitric oxide homeostasis within the guard cells of AOX knockdown plants (Cvetkovska et al., 2014).Drought is a common abiotic stress that can substantially curtail photosynthesis because stomatal closure, meant to conserve water, also restricts CO₂ availability to the Calvin cycle. Besides this well established stomatal limitation of photosynthesis, there may also be water deficit-sensitive biochemical components that contribute to the reduction of photosynthesis during drought. However, the nature of this biochemical limitation and the degree to which it contributes to the curtailment of photosynthesis during drought remain areas of active debate (Flexas et al., 2004; Lawlor and Tezara, 2009; Pinheiro and Chaves, 2011). Additional factors, such as patchy stomatal closure (Sharkey and Seemann, 1989; Gunasekera and Berkowitz, 1992) or changes in the conductance to CO₂ of mesophyll cells (Perez-Martin et al., 2009), can further complicate analyses of photosynthesis during drought.Metabolism can experience energy imbalances, when there is a mismatch between rates of synthesis and rates of utilization of ATP and/or NADPH, and the importance of mechanisms to minimize such imbalances has been emphasized (Cruz et al., 2005; Kramer and Evans, 2011; Vanlerberghe, 2013). For example, such imbalances may occur in the chloroplast when the use of ATP and NADPH by the Calvin cycle does not keep pace with the harvesting of light energy (Hüner et al., 2012). This can result in excess excitation energy that can damage photosynthetic components, perhaps through the generation of ROS (Asada, 2006; Noctor et al., 2014). Such a scenario has been hypothesized to underlie the development of the biochemical limitations of photosynthesis reported during drought (Lawlor and Tezara, 2009).In this study, we find that N. tabacum AOX knockdowns show a compromised rate of mitochondrial respiration in the light during moderate drought. This corresponds with a strong nonstomatal limitation of photosynthesis in these plants relative to the wild type, and we describe a biochemical basis for this photosynthetic limitation. The results indicate that AOX is a necessary electron sink to support photosynthesis during drought, a condition when the major photosynthetic electron sink, the Calvin cycle, is becoming limited by CO₂ availability.