Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules.

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.

Immunoglobulin G1 Antibodies Against Phosphorylcholine Are Associated With Protection in Systemic Lupus Erythematosus and Atherosclerosis: Potential Underlying Mechanisms.

OBJECTIVE: Immunoglobulin M antibodies against phosphorylcholine (anti-PCs) may be protective in atherosclerosis, cardiovascular disease (CVD), and systemic lupus erythematosus (SLE). We study immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) anti-PCs, with a focus on atherosclerosis and SLE. METHODS: We determined anti-PCs by using the enzyme-linked immunosorbent assay in 116 patients with SLE and 110 age- and sex-matched controls. For functional studies, we used three in-house-generated, fully human monoclonal IgG1 anti-PCs (A01, D05, and E01). Apoptosis was induced in Jurkat T cells and preincubated with A01, D05, E01, or IgG1 isotype control, and effects on efferocytosis by human macrophages were studied. Anti-PC peptide/protein characterization was determined using a proteomics de novo sequencing approach. RESULTS: IgG1, but not IgG2, anti-PC levels were higher among patients with SLE (P = 0.02). IgG1 anti-PCs were negatively associated with Systemic Lupus International Collaborating Clinics (SLICC) damage index and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores (odds ratio [OR]: 2.978 [confidence interval (CI): 0.876-10.098] and OR: 5.108 [CI 1.3-20.067], respectively) and negatively associated with CVD, atherosclerotic plaques, and echolucent plaques (potentially vulnerable plaques), but the association for the two former was not significant after controlling for confounders. D05 had a maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC-associated neoepitopes. A peptide analysis showed a difference in the complementarity-determining region 3 of the three IgG1 anti-PC clones that are crucial for recognition of PC on apoptotic cell surfaces and other neoepitopes. CONCLUSION: IgG1 anti-PCs are negatively associated with disease activity and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells.

Autoantibody profiling reveals four protein candidate autoantigens associated with systemic lupus erythematosus.

Objectives In systemic lupus erythematosus (SLE) there are typically many autoantibodies. The disease heterogeneity could be better understood with discovery of phenotype-specific antigens targeted by autoantibodies. We here aimed to identify novel autoantigens potentially related to SLE disease and a major complication, atherosclerosis. Methods Antigen microarrays were used to profile IgG autoantibody reactivity against 77 protein fragments (20-140 amino acids (aa) long, median 89 aa) produced within the Human Protein Atlas project, in serum samples from SLE patients ( n = 107) and age- and sex-matched population-based controls ( n = 107). Common carotid intima-media thickness, plaque occurrence and echogenicity were determined by B-mode ultrasound. Results We determined significant differences between patients and controls in IgG reactivity against four proteins. In patients compared to controls, there was an increase of IgG reactivity against zinc finger protein 688 (ZNF688), early B cell factor 2 (EBF2), crystallin, alpha B (CRYAB) and tumor necrosis factor receptor superfamily member 13C (TNFRSF13C). Of these four antigens, only anti-ZNF688 was associated with carotid atherosclerosis (plaque occurrence) and vulnerable plaques in SLE. There was a weak association between anti-EBF2 and SLE disease activity but no significant associations were determined for other measured IgG reactivity. Conclusions In this discovery screening we here demonstrate new candidate autoantigens with differential reactivity (reflecting autoantibody levels) in SLE patients and in controls and in relation to atherosclerosis in SLE.

Carbon-driven enrichment of the crucial nitrate-reducing bacteria in limed peat soil microcosms.

Bacteria of Dechloromonas were recognized as potential functional important denitrifiers in a long-term shell sand-amended peat soil. Different microcosms in a solid matrix and slurry systems with the addition of carbon and nitrogen sources, for example, clover leaves, glutamate and nitrate, were established. The bacterial community structures were analysed by pyrosequencing of the 16S rRNA gene to select the conditions for enriching bacteria of Dechloromonas. The results showed that a relatively even bacterial community in the initial soil shifted to communities dominated by a few types of nitrate-reducing bacteria after the incubation, which strongly responded to the carbon substrates addition and consumption. The bacteria of several genera including Dechloromonas, Pseudomonas, Clostridium, Aeromonas and Ferribacterium were significantly enriched after a certain period of time. The bacteria of Dechloromonas became one of the most predominant bacteria in the incubated community. Especially when added the mixed carbon substrates into the solid soil matrix, as high as 34% of abundance was detected. This study proved that the functional important bacteria from the genus of Dechloromonas could be enriched to an extremely high abundance by using proper culture condition which will benefit to the isolation or direct metagenomics study for Dechloromonas. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of key players in a microbial community is always of important. In this study, the functional important denitrifiers in a shell sand-amended peat soil were investigated. Using different carbon sources in the incubation, we found the bacteria from the genus of Dechloromonas were enriched to an abundance of higher than 34% with several other denitrifiers together. This work provides us helpful insights not only for knowing the diversity of denitrifiers in the studied peat soil, but also for understanding their response to the carbon sources and the culture conditions.

Immunoglobulin (Ig)M antibodies against oxidized cardiolipin but not native cardiolipin are novel biomarkers in haemodialysis patients, associated negatively with mortality.

The risk of premature death is high in haemodialysis (HD) patients. Antibodies against cardiolipin (anti-CL) are thrombogenic in diseases such as systemic lupus erythematosus (SLE). CL is easily oxidized (Ox) and plays a role in apoptosis. In this work we studied immunoglobulin (Ig)M anti-CL and anti-OxCL in HD-patients. We conducted an observational study with a prospective follow-up examining the relationship between anti-CL, anti-OxCL and mortality risk in a well-characterized cohort of 221 prevalent HD patients [56% men, median age 66 (interquartile range 51-74) years, vintage time 29 (15-58) months] with a mean follow-up period of 41 (20-48 months). According to the receiver operator characteristic (ROC) analysis, anti-OxCL [area under the curve (AUC) 0·62, P < 0·01], but not anti-CL (AUC 0·52, P = 0·2), is associated with mortality. In crude and adjusted Cox analysis, every log increase in anti-OxCL inversely predicted all-cause [adjusted hazard ratios (HR) 0·62 (0·43-0·89)] and CVD-related [adjusted HR 0·56 (0·32-0·98)] mortality. Patients with anti-OxCL levels below median also had increased all-cause and cardiovascular disease (CVD)-related mortality. Although anti-OxCL and anti-phosphorylcholine (PC) were related positively to each other (ρ = 0·57, P < 0·01), patients with one or two of these autoantibody levels below the median were associated with an incrementally increased death risk. Anti-OxCL were co-factor ß2-GPI-independent; anti-CL from patients with anti-phospholipid antibody syndrome were ß2-GPI-dependent, while sera from HD-patients less so. Sera from healthy donors was not ß2-GPI-dependent. Anti-OxCL IgM is ß2-glycoprotein 1 (GPI)-independent and a novel biomarker; low levels are associated with death among HD patients (and high levels with decreased risk). Combination with anti-PC increases this association. Putative therapeutic implications warrant further investigation.

Effects of anti-cardiolipin antibodies and IVIg on annexin A5 binding to endothelial cells: implications for cardiovascular disease.

OBJECTIVES: Anti-phospholipid antibodies (aPL), including anti-cardiolipin antibodies (aCL), are risk factors for cardiovascular disease (CVD) in the general population and in patients with the anti-phospholipid syndrome (APS; Hughes syndrome). APS may be primary but is also common in patients with systemic lupus erythematosus (SLE). The anti-coagulant protein annexin A5 (ANXA5) is implicated in CVD by interfering with phospholipids and aPL. METHODS: ANXA5 binding to human umbilical venous endothelial cells (HUVECs) was determined by flow cytometry. RESULTS: When cells were cultured in serum from APS patients with a high aPL titre (aPL-S), binding of ANXA5 to HUVECs was reduced. Monoclonal immunoglobulin (Ig)G aPL against cardiolipin (mAb-CL) dose-dependently reduced ANXA5 binding to endothelium. Preincubation of intravenous (IV)Ig at therapeutically relevant doses with aPL-S and mAb-aCL restored ANXA5 binding to comparable levels when normal healthy serum (NHS) was used. By contrast, IVIg per se had the capacity to reduce ANXA5 binding to endothelium when added to NHS (but not to aPL-S). CONCLUSIONS: Decreased ANXA5 binding to endothelium, mediated by aPL, is a novel mechanism of atherothrombosis that can be countered by IVIg in vitro. IVIg per se could, to a lesser degree, cause decreased ANXA5 binding in NHS, which raises the possibility that some antibodies in IVIg can be involved in a side-effect reported in IVIg treatment, namely atherothrombosis and CVD. Increasing ANXA5 binding, either by addition of ANXA5 or by use of neutralizing antibodies in IVIg, represents a possible therapeutic strategy that deserves further study.

Retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters.

AIMS: To investigate the retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters and effects on the microbial community composition. METHODS AND RESULTS: Sand filter columns were loaded (70 mm day(-1)) with fish farm wastewater and a suspension (10(8) CFU ml(-1)) of Y. ruckeri. Bacterial numbers and protozoan numbers were determined by plate counts and epifluorescence microscopy, respectively, and microbial biomass and community composition were assessed by phospholipid fatty acids (PLFA) analysis. Concentrations of Y. ruckeri in the filter effluent decreased from 10(8) to 10(3)-10(5) CFU ml(-1) during the experiment. Numbers of Y. ruckeri in the sand decreased from 10(6) CFU g(-1) dry weight (DW) sand to 10(4) CFU g(-1) DW sand. In contrast, microbial biomass determined with plate counts and total PLFA increased during the whole experiment. Principal component analysis (PCA) revealed a change in microbial community composition with time, with the most pronounced change in surface layers and towards the end of the experiment. Protozoan numbers increased from ca 0-600 cells g(-1) DW sand, indicating the establishment of a moderate population of bacterial grazers. CONCLUSIONS: The removal of Y. ruckeri improved during the experiment. Introduction of Y. ruckeri to the sand filter columns stimulated growth of other micro-organisms, which in turn caused a shift in the microbial community composition in the sand. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the understanding of the dynamics of sand filters subjected to a high loading of a pathogenic bacterium and can therefore be used in future work were the overall aim is to provide a more reliable and efficient removal of pathogenic bacteria in biological sand filter systems.

Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation.

All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 microg g(-1) dry weight soil versus 38-93 microg g(-1) obtained by in situ lysis methods). However, with the exception of the gamma-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies.

Quantification of bias related to the extraction of DNA directly from soils.

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage lambda DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

Responses of the soil microbiota to elevated CO2 in an artificial tropical ecosystem.

Plants in artificial tropical ecosystems were grown under ambient (340 microl l(-1)) and elevated (610 microl l(-1)) atmospheric CO2 for 530 d under low-nutrient conditions on a substrate free of organic C. At the end of the experiment a number of soil chemical and microbiological variables were determined. Although we found no changes in total soil organic matter under elevated CO2, we did find that after physical fractionation the amount of organic C in the supernatant (< 0.2 microm) and the amount of water extractable organic C (WEOC) was lower under elevated CO2. The extractable optical density (OD) indicated a higher degree of humification for the elevated than for the ambient CO2 samples (P = 0.032). Microbial biomass C was not significantly altered under high CO2, but total bacterial counts were significantly higher. The microbial biomass C-to-N ratio was also higher at elevated (15.0) than at ambient CO2 (10.0). The number of mycorrhizal spores was lower at high CO2, but ergosterol contents and fungal hyphal lengths were not significantly affected. Changes were found neither in community level physiological profiles (CLPPs) nor in the structural attributes (phospholipid fatty acids, PLFAs) of the microbial community. Overall, the effects on the soil microbiota were small, perhaps as a result of the low nutrient supply and low organic matter content of the soil used in our study. The few significant results showing changes in specific, though relatively minor, organic matter pools may point to possible long-term changes of the more major pools. Furthermore, the data suggest increased competition between plants and microbes for N at high CO2.

Dynamics of a microbial community associated with manure hot spots as revealed by phospholipid fatty acid analyses.

Microbial community dynamics associated with manure hot spots were studied by using a model system consisting of a gel-stabilized mixture of soil and manure, placed between layers of soil, during a 3-week incubation period. The microbial biomass, measured as the total amount of phospholipid fatty acids (PLFA), had doubled within a 2-mm distance from the soil-manure interface after 3 days. Principal-component analyses demonstrated that this increase was accompanied by reproducible changes in the composition of PLFA, indicating changes in the microbial community structure. The effect of the manure was strongest in the 2-mm-thick soil layer closest to the interface, in which the PLFA composition was statistically significantly different (P < 0.05) from that of the unaffected soil layers throughout the incubation period. An effect was also observed in the soil layer 2 to 4 mm from the interface. The changes in microbial biomass and community structure were mainly attributed to the diffusion of dissolved organic carbon from the manure. During the initial period of microbial growth, PLFA, which were already more abundant in the manure than in the soil, increased in the manure core and in the 2-mm soil layer closest to the interface. After day 3, the PLFA composition of these layers gradually became more similar to that of the soil. The dynamics of individual PLFA suggested that both taxonomic and physiological changes occurred during growth. Examples of the latter were decreases in the ratios of 16:1 omega 7t to 16:1 omega 7c and of cyclopropyl fatty acids to their respective precursors, indicating a more active bacterial community. An inverse relationship between bacterial PLFA and the eucaryotic 20:4 PLFA (arachidonic acid) suggested that grazing was important.

Phospholipid Fatty Acid Composition and Heavy Metal Tolerance of Soil Microbial Communities along Two Heavy Metal-Polluted Gradients in Coniferous Forests.

The effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community tolerance were studied along two different gradients in Scandinavian coniferous forest soils. One was near the Harjavalta smelter in Finland, and one was at Ronnskar in Sweden. Phospholipid fatty acid (PLFA) analysis revealed a gradual change in soil microbial communities along both pollution gradients, and most of the individual PLFAs changed similarly to metal pollution at both sites. The relative quantities of the PLFAs br18:0, br17:0, i16:0, and i16:1 increased with increasing heavy metal concentration, while those of 20:4 and 18:2(omega)6, which is a predominant PLFA in many fungi, decreased. The fungal part of the microbial biomass was found to be more sensitive to heavy metals. This resulted in a decreased fungal/bacterial biomass ratio along the pollution gradient towards the smelters. The thymidine incorporation technique was used to study the heavy metal tolerance of the bacteria. The bacterial community at the Harjavalta smelter, exposed mainly to Cu deposition, exhibited an increased tolerance to Cu but not to Cd, Ni, and Zn. At the Ronnskar smelter the deposition consisting of a mixture of metals increased the bacterial community tolerance to all tested metals. Both the PLFA pattern and the bacterial community tolerance were affected at lower soil metal concentrations than were bacterial counts and bacterial activities. At Harjavalta the increased Cu tolerance of the bacteria and the change in the PLFA pattern of the microbial community were found at the same soil Cu concentrations. This indicated that the altered PLFA pattern was at least partly due to an altered, more metal-tolerant bacterial community. At Ronnskar, where the PLFA data varied more, a correlation between bacterial community tolerance and an altered PLFA pattern was found up to 10 to 15 km from the smelter. Farther away changes in the PLFA pattern could not be explained by an increased community tolerance to metals.

Multiple heavy metal tolerance of soil bacterial communities and its measurement by a thymidine incorporation technique.

A thymidine incorporation technique was used to determine the tolerance of a soil bacterial community to Cu, Cd, Zn, Ni, and Pb. An agricultural soil was artificially contaminated in our laboratory with individual metals at three different concentrations, and the results were compared with the results obtained by using the plate count technique. Thymidine incorporation was found to be a simple and rapid method for measuring tolerance. Data obtained by this technique were very reproducible. A linear relationship was found between changes in community tolerance levels obtained by the thymidine incorporation and plate count techniques (r = 0.732, P < 0.001). An increase in tolerance to the metal added to soil was observed for the bacterial community obtained from each polluted soil compared with the community obtained from unpolluted soil. The only exception was when Pb was added; no indication of Pb tolerance was found. An increase in the tolerance to metals other than the metal originally added to soil was also observed, indicating that there was multiple heavy metal tolerance at the community level. Thus, Cu pollution, in addition to increasing tolerance to Cu, also induced tolerance to Zn, Cd, and Ni. Zn and Cd pollution increased community tolerance to all five metals. Ni amendment increased tolerance to Ni the most but also increased community tolerance to Zn and, to lesser degrees, increased community tolerance to Pb and Cd. In soils polluted with Pb increased tolerance to other metals was found in the following order: Ni > Cd > Zn > Cu. We found significant positive relationships between changes in Cd, Zn, and Pb tolerance and, to a lesser degree, between changes in Pb and Ni tolerance when all metals and amendment levels were compared. The magnitude of the increase in heavy metal tolerance was found to be linearly related to the logarithm of the metal concentration added to the soil. Threshold tolerance concentrations were estimated from these linear relationships, and changes in tolerance could be detected at levels of soil contamination similar to those reported previously to result in changes in the phospholipid fatty acid pattern (A. Frostegård, A. Tunlid, and E. Bååth, Appl. Environ. Microbiol. 59: 3605-3617, 1993).

Phospholipid Fatty Acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals.

The phospholipid fatty acid (PLFA) pattern was analyzed in a forest humus and in an arable soil experimentally polluted with Cd, Cu, Ni, Pb, or Zn at different concentrations. In both soil types, there were gradual changes in the PLFA patterns for the different levels of metal contamination. The changes in the forest soil were similar irrespective of which metal was used, while in the arable soil the changes due to Cu contamination differed from those due to the other metals. Several PLFAs reacted similarly to the metal amendments in the two soil types, while others showed different responses. In both soils, the metal pollution resulted in a decrease in the iso-branched PLFAs i15:0 and i17:0 and in the monounsaturated 16:1omega5 and 16:1omega7c fatty acids, while increases were found for i16:0, the branched br17:0 and br18:0, and the cyclopropane cy17:0 fatty acids. In the forest soil, the methyl branched PLFAs 10Me16:0, 10Me17:0, and 10Me18:0 increased in metal-polluted soils, indicating an increase in actinomycetes, while in the arable soil a decrease was found for 10Me16:0 and 10Me18:0 in response to most metals. The bacterial PLFAs 15:0 and 17:0 increased in all metal-contaminated samples in the arable soil, while they were unaffected in the forest soil. Fatty acid 18:2omega6, which is considered to be predominantly of fungal origin, increased in the arable soil, except in the Cu-amended samples, in which it decreased instead. Effects on the PLFA patterns were found at levels of metal contamination similar to or lower than those at which effects on ATP content, soil respiration, or total amount of PLFAs had occurred.

Soil Bacterial Biomass, Activity, Phospholipid Fatty Acid Pattern, and pH Tolerance in an Area Polluted with Alkaline Dust Deposition.

Soil bacterial biomass, phospholipid fatty acid pattern, pH tolerance, and growth rate were studied in a forest area in Finland that is polluted with alkaline dust from an iron and steel works. The pollution raised the pH of the humus layer from 4.1 to 6.6. Total bacterial numbers and the total amounts of bacterial phospholipid fatty acids in the humus layer did not differ between the unpolluted control sites and the polluted ones. The number of CFU increased by a factor of 6.4 in the polluted sites compared with the controls, while the bacterial growth rate, measured by the thymidine incorporation technique, increased about 1.8-fold in the polluted sites. A shift in the pattern of phospholipid fatty acids indicated a shift in the bacterial species composition. The largest proportional increase was found for the fatty acid 10Me18:0, which indicated an increase in the number of actinomycetes in the polluted sites. The levels of the fatty acids i14:0, 16:1omega5, cy17:0, 18:1omega7, and 19:1 also increased in the polluted sites while those of fatty acids 15:0, i15:0, 10Me16:0, 16:1omega7t, 18:1omega9, and cy19:0 decreased compared with the unpolluted sites. An altered pH tolerance of the bacterial assemblage was detected either as a decrease in acid-tolerant CFU in the polluted sites or as altered bacterial growth rates at different pHs. The latter was estimated by measuring the thymidine incorporation rate of bacteria extracted from soil by homogenization-centrifugation at different pHs.