Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.     

The contribution of nitrogen by promiscuous soybeans to maize based cropping the moist savanna of Nigeria

Agronomic results indicate that maize grain yields generally are higher when the crop is planted following soybean than in continuous maize cultivation in the moist savanna agroecological zones of West Africa. Many factors have been hypothesized to explain this phenomenon, including enhanced N availability and the so-called `rotational effect'. There is, however, hardly any quantitative information on the residual N benefits of promiscuous soybeans to subsequent cereal crops grown in rotation with soybean. Three IITA promiscuous soybean breeding lines and two Brazilian soybean lines were grown in 1994 and 1995 at Mokwa in the southern Guinea savanna, Nigeria, to quantify the nitrogen contribution by soybeans to a succeeding crop of maize grown in rotation with soybean for two consecutive years, 1996 and 1997 using two methods of introducing ¹⁵N into soil (fresh ¹⁵N labelling and its residual ¹⁵N) and three maize cultivars (including one cultivar with high N use efficiency) used as reference plants. The nodulating soybeans fixed between 44 and 103 kg N ha^–1 of their total N and had an estimated net N balance input from fixation following grain harvest ranging from –8 to 43 kg N ha^–1. Results in 1996 and in 1997 showed that maize growing after soybean had significantly higher grain yield (1.2 – 2.3-fold increase compared to maize control) except for maize cultivar Oba super 2 (8644-27) (a N-efficient hybrid). The ¹⁵N isotope dilution method was able to estimate N contribution by promiscuous soybeans to maize only in the first succeeding maize crop grown in 1996 but not in the second maize crop in 1997. The first crop of maize grown after soybean accumulated an average between 10 and 22 kg N ha^–1 from soybean residue, representing 17–33% of the soybean total N ha^–1. The percentage ¹⁵N derived from residue recovery in maize grown after maize was influenced by the maize cultivars. Maize crop grown after the N-efficient hybrid cultivar Oba Super 2 (844-27) had similar ¹⁵N values similar to maize grown after soybeans, confirming the ability of this cultivar to use N efficiently in low N soil due to an efficient N translocation ability. The maize crop in 1997 grown after maize had lower ¹⁵N enrichment than that grown in soybean plots, suggesting that soybean residues contributed a little to soil available N and to crop N uptake by the second maize crop. The differential mineralization and immobilization turnover of maize and soybean residues in these soils may be important and N contribution estimates in longer term rotation involving legumes and cereals may be difficult to quantify using the ¹⁵N labelling approaches. Therefore alternative methods are required to measure N release from organic residues in these cropping systems.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

The effect of lincomycin on the excretion of diethylstilbestrol and its uterotrophic action in rats.

The enterohepatic circulation of diethylstilbestrol (DES) has been shown to be extensive and to be dependent on enteric β-glucuronidase activity for release of absorbable DES from its nonabsorbable glucuronide excreted in bile. A regimen of the antibiotic lincomycin has been shown in rats to markedly reduce enteric β-glucuronidase activity, intestinal hydrolysis of C¹⁴-DES-glucuronide and absorption of radioactivity. Studies were therefore performed to determine if this lincomycin regimen, by reducing enterohepatic circulation of DES, would alter systemic effects of DES such as uterine weight gain in ovariectomized rats. The lincomycin regimen consisted of 25 mg twice daily by gastric intubation on days 1–4 and 500 mg/l in drinking water on days 1–7. Lincomycin-treated and control rats were injected s.c. with DES (1.6 or 5.0 μg/kg/day) on days 4–6 and sacrificed on day 7 for measurement of uterine weight; the injection on day 4 consisted on monoethyl-l-C¹⁴-DES. Lincomycin did not change the uterotrophic effect of DES. The regimen did, however, reduce the urinary excretion of radioactivity and increase the fecal excretion of glucuronide conjugates, consistent with reduced enterohepatic circulation of DES. In a separate study, bile from rats given DES s.c. was administered into the cecum of lincomycin-treated and control rats; whereas 31.8% of the cecal dose was excreted in the bile of controls, only 1.9% was excreted in lincomycin-treated rats, indicating the marked reduction of DES enterohepatic circulation produced by lincomycin. These findings suggest that the enterohepatic circulation of DES does not significantly contribute to its systemic effects.     

Assessing larval rootworm behaviour after contacting maize roots: impact of germplasm,rootworm species and diapause status

Current methods of screening maize (Zea mays L.) germplasm for susceptibility or resistance to corn rootworms (Coleoptera: Chrysomelidae) rely primarily on information from large‐scale field experiments. Due to labour and cost constraints associated with field trials, alternative evaluation methods are desirable. We used a previously developed behavioural bioassay to: (1) investigate the host search behaviour of rootworm larvae after contact with 14 maize genotypes, (2) compare the behaviour of non‐diapausing Diabrotica virgifera virgifera LeConte, diapausing D. v. virgifera, and diapausing D. barberi Smith & Lawrence and (3) determine if this technique can be used to separate susceptible vs. resistant maize genotypes. The majority of rootworm larvae engaged in intensive (local search) behaviour after exposure to maize roots, whereas larvae continued to exhibit extensive (ranging) behaviour after contact with negative controls. Even though a transgenic hybrid with resistance to D. v. virgifera was included in analyses, quantitative path measurements were similar among genotypes and only differed between specific maize lines and controls. Notably, there were differences in host search behaviour among rootworm groups, with non‐diapausing D. v. virgifera having more convoluted paths and engaging in intensive search more frequently than diapausing rootworms. Correlations between larval path measurements and historic root damage ratings were not significant, although there were weak positive correlations between historic adult emergence densities and measures of path linearity. However, due to the lack of significant behavioural differences among maize lines with a range of susceptibility levels, we concluded that this bioassay is not useful in screening maize germplasm for rootworm resistance.     

Field and laboratory evaluations of soybean lines against soybean aphid (Hemiptera: Aphididae)

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a major pest of soybean, Glycine max (L.). Merr., that significantly reduces yield in northern production areas of North America. Insecticides are widely used to control soybean aphid outbreaks, but efforts are underway to develop host plant resistance as an effective alternative management strategy. Here, previously identified resistant lines were evaluated in laboratory tests against field-collected populations of soybean aphid and in field-plot tests over 2 yr in South Dakota. Six lines previously identified with resistance to soybean aphid--Jackson, Dowling, K1639, Cobb, Palmetto and Sennari--were resistant in this study, but relatively high aphid counts on Tie-feng 8 in field plots contrasted with its previously reported resistance. Bhart-PI 165989 showed resistance in one of two laboratory tests, but it had relatively large aphid infestations in both years of field tests. Intermediate levels of soybean aphid occurred in field plots on lines previously shown to have strong (Sugao Zairai, PI 230977, and D75-10169) or moderate resistance to soybean aphid (G93-9223, Bragg, Braxton, and Tracy-M). Sugao Zairai also failed to have a significant proportion of resistant plants in two laboratory tests against aphids field-collected in 2008, but it was resistant in laboratory tests with aphids collected in 2002, 2005, and 2006. Overall, results showed that lines with Rag (i.e., Jackson) or Rag1 gene (i.e., Dowling) had low aphid numbers, whereas lines with Rag2 (i.e., Sugao Zairai, Sennari) had mixed results. Collectively, responses of soybean aphid populations in laboratory and field tests in 2008 resembled a virulence pattern reported previously for biotype 3 soybean aphids, but virulence in soybean aphid populations was variable and dynamic over years of the study. These results, coupled with previous reports of biotypes virulent to Rag1, suggest that deployment of lines with a single aphid-resistance gene is limited for soybean aphid management, and that deployment strategies relying on multiple resistance genes may be needed to effectively use plant resistance against soybean aphid.     

Fate of the porphyrin cofactors during the light-dependent turnover of catalase and of the photosystem II reaction-center protein D1 in mature rye leaves

The apoprotein of the enzyme catalase (EC 1.11.1.6) was shown to exhibit a light-dependent turnover in leaves. Present results indicate that photoinactivation of the enzyme was not accompanied by a synchronous destruction and new synthesis of its heme moiety. In rye (Secale cereale L.) leaves the catalase content was not depleted in light when porphyrin synthesis was inhibited by gabaculine. Photoinactivation of purified bovine liver or rye leaf catalase in vitro was not accompanied by concomitant damage to the heme groups. Both the incorporation of -[³H]aminolevulinic acid ([³H]ALA) into catalase-heme and its apparent turnover increased with irradiance. However, the apparent half-life of the catalase-heme was much longer than that of its apoprotein. It is probable that not only degradation but also an exchange with the free heme pool contributed to the apparent turnover of radioactivity of the catalase-heme. Part of the chlorophyll (Chl) associated with photosystem II (PS II) had a preferential light-induced turnover, and repair of PS II appeared to require new Chl synthesis also in mature green rye leaves. The activity of PS II, indicated by the ratio of variable to maximal fluorescence (F_v/F_m), rapidly declined in the presence of gabaculine in light and the reaction-center proteins D1 and D2 were depleted. When segments of mature green rye leaves were labeled with [³H]ALA and incorporation into Chl-protein complexes analysed after electrophoretic separation in the presence of Deriphat, the highest radioactivity was observed in the core complex of PS II, while PS I and the light-harvesting complex of PS II (LHC II) were unlabeled. In greening etiolated leaves highest incorporation was observed in LHC II. Both the incorporation of [³H]ALA into the PS II core complex of green rye leaves and its turnover increased with irradiance. However, the apparent half-life of the PS II-bound labeled porphyrin compounds (mainly Chl) was considerably longer than that of the reaction-center protein D1 under identical conditions.Abbreviations ALA -aminolevulinic acid - CII Core complex of PS II - Chl chlorophyll - DMSO dimethyl sulfoxide - F_v/F_m ratio of variable to maximal chlorophyll fluorescence - LHC light-harvesting complex - PAR photosynthetically active radiation We thank the Deutsche Forschungsgemeinschaft for financial support. Technical assistence by B. Kramer and Ch. van Oijen is greatly appreciated. We are grateful to Dr. Johanningmeier and Dr. Godde (Lehrstuhl für Biochemie der Pflanzen, Universität Bochum, Germany) for providing antisera against the D1 and D2 proteins and Dr. M. Schmidt (Botanisches Institut, Universität Frankfurt am Main, Germany) for valuable advice. Deriphat 160 was kindly supplied by Henkel Corp., Hoboken, N.J., USA.     

Divergent strategies of photoprotection in high-mountain plants

Leaves of high-mountain plants were highly resistant to photoinhibitory damage at low temperature. The roles of different photoprotective mechanisms were compared. Mainly, the alpine species Ranunculus glacialis (L.) and Soldanella alpina were investigated because they appeared to apply greatly divergent strategies of adaptation. The ratio of electron transport rates of photosystem II/photosystem I measured in thylakoids from R. glacialis did not indicate a specific acclimation to high irradiance. Low rates of a chloroplast-mediated inactivation of catalase (EC 1.11.1.6) in red light indicated, however, that less reactive oxygen was released by isolated chloroplasts from R. glacialis than by chloroplasts from lowland plants. Leaves of S. alpina and of Homogyne alpina (L.) Cass, but not those of R. glacialis, had a very high capacity for antioxidative protection, relative to lowland plants, as indicated by a much higher tolerance against paraquat-mediated photooxidative damage and a higher -tocopherol content. Accordingly, ascorbate and glutathione were strongly oxidized and already largely destroyed at low paraquat concentrations in leaves of R. glacialis, but were much less affected in leaves of  S. alpina. Non-radiative dissipation of excitation energy was essential for photoprotection of leaves of  S. alpina and depended on the operation of the xanthophyll cycle. Strong non-photochemical quenching of chlorophyll fluorescence occurred also in R. glacialis leaves at high irradiance, but was largely independent of the presence of zeaxanthin or antheraxanthin. For R. glacialis, photorespiration appeared to provide a strong electron sink and a most essential means of photoprotection, even at low temperature. Application of phosphinothricin, which interferes with photorespiration by inhibition of glutamine synthetase, caused a striking reduction of electron transport through photosystem II and induced marked photoinhibition at both ambient and low temperature in leaves of R. glacialis, while  S. alpina was less affected. Received: 18 March 1998 / Accepted: 7 August 1998     

Significance of antioxidants and electron sinks for the cold-hardening-induced resistance of winter rye leaves to photo-oxidative stress

The contents of ascorbate and glutathione and the activities of superoxide dismutase and glutathione reductase were increased to levels as high as those in cold-hardened leaves (CHL) by incubating non-hardened leaves (NHL) of winter rye (Secale cereale L.) with the precursor substrates L -galactonic acid-γ-lactone and 2-oxothiazolidine-4-carboxylate. Reduced glutathione was rapidly depleted from NHL after application of D , L -buthionine sulfoximine, an inhibitor of its biosynthesis. In spite of greatly divergent antioxidant contents the rates of photo-inactivation of photosystem II (PSII) and catalase observed in the presence of translation inhibitors did not differ greatly. The paraquat-induced catalase inactivation and chlorophyll degradation in light were reduced in NHL with increased antioxidant levels. Paraquat-induced photo-inactivation of PSII was, however, not mitigated. The CHL had a higher capacity to prevent paraquat-induced oxidation of ascorbate and glutathione than NHL with increased antioxidant contents. Increased antioxidant contents did not establish resistance to low temperature-induced photo-inactivation of PSII and catalase in NHL. The resistance of CHL to low temperature-induced photo-inactivation of PSII and catalase required repair at low temperature and active carbon assimilation but was only little affected when photorespiration was suppressed by phosphinothricin. Protection of PSII depended also on non-photochemical quenching of excitation energy.