Identification of the Corn Pathogen Pantoea stewartii by Mass Spectrometry of Whole-Cell Extracts and Its Detection with Novel PCR Primers

Pantoea stewartii subsp. stewartii is the causative agent of Stewart''s wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn (Zea mays). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans, Pantoea dispersa, and Pantoea ananatis. Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA.Stewart''s wilt, caused by Pantoea stewartii subsp. stewartii (synonym Erwinia stewartii) is a serious disease of sweet corn (Zea mays) that was originally described in the United States (17, 18). Its transmission depends on the corn flea beetle (Chaetocnema pulicaria), which ingests the pathogen from infected tissue and transfers the bacteria to healthy plants. The beetle is also the main niche for overwintering of P. stewartii. Direct distribution by seed transmission is also possible (3, 11) but is not considered a major source. Stewart''s wilt is also a problem on certain elite inbred maize lines used for producing hybrid field corn seed in the mideastern United States (2). According to data from the European and Mediterranean Plant Protection Organization (EPPO) about its occurrence in Europe, Stewart''s wilt was reported from but not established in Austria, Greece, Poland, Romania, and Russia. More than 60 countries place import regulations on maize seed imports from affected areas, and surveillance of traded plant material is required to prevent further distribution of the pathogen (14).Several detection methods have been described for P. stewartii, including monoclonal antibodies for enzyme-linked immunosorbent assay (ELISA) (8). For the detection of P. stewartii by PCR analysis, primer pairs derived from rRNA genes and chromosomal markers, such as regions coding for the Hrp type III secretion system (hrp) and capsular exopolysaccharide (EPS) synthesis (cps), have been published (4). These primers were derived from chromosomal regions which are also common to other bacteria. A unique DNA area of P. stewartii might therefore be better suited for the design of specific primers. Another approach, the ligase chain reaction, requires radioactively labeled primers (21). Primers complementary to cpsD (wceL) were applied for quantitative PCR (qPCR) (19). A fingerprinting analysis based on miniprimer PCR and utilizing 10-mer short oligonucleotides combined with modified Taq polymerase has been reported (22). The signal intensity of PCRs is often affected by inhibitory plant components in the extracts. Thus, low levels of P. stewartii may not be detected. A collective drawback of PCR-based identification approaches is the detection of DNA from nonviable cells and traces of residual nucleic acids. This could lead to the rejection of safe seed lots. A method involving culturing of bacteria extracted from plants, lysis, and subsequent PCR analysis and named bio-PCR was established to ensure the detection of only viable bacterial populations (16). Screening of individual colonies from a plate with mixed cultures by PCR to verify reisolation of the pathogen is tedious and needs another fast and reliable method. Strains of Pantoea stewartii subsp. indologenes cause leaf spot on foxtail millet (Setaria italica) and pearl millet (Pennisetum americanum) or rot of Ananas comosus, and one strain was isolated from a diseased cluster bean (Cyamopsis tetragonolobus) (10). It is important to distinguish between P. stewartii subsp. stewartii and P. stewartii subsp. indologenes, since only the stewartii subspecies causes Stewart''s wilt.Furthermore, some bacterial isolates might not be unambiguously identified with PCR and with phytopathological methods. The recent successful identification of Erwinia isolates with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis profiling of protein patterns from whole cells (15) induced us to apply this method for the detection of P. stewartii and its differentiation from Pantoea agglomerans and other Pantoea species.     

Weathering-Associated Bacteria from the Damma Glacier Forefield: Physiological Capabilities and Impact on Granite Dissolution

Several bacterial strains isolated from granitic rock material in front of the Damma glacier (Central Swiss Alps) were shown (i) to grow in the presence of granite powder and a glucose-NH₄Cl minimal medium without additional macro- or micronutrients and (ii) to produce weathering-associated agents. In particular, four bacterial isolates (one isolate each of Arthrobacter sp., Janthinobacterium sp., Leifsonia sp., and Polaromonas sp.) were weathering associated. In comparison to what was observed in abiotic experiments, the presence of these strains caused a significant increase of granite dissolution (as measured by the release of Fe, Ca, K, Mg, and Mn). These most promising weathering-associated bacterial species exhibited four main features rendering them more efficient in mineral dissolution than the other investigated isolates: (i) a major part of their bacterial cells was attached to the granite surfaces and not suspended in solution, (ii) they secreted the largest amounts of oxalic acid, (iii) they lowered the pH of the solution, and (iv) they formed significant amounts of HCN. As far as we know, this is the first report showing that the combined action of oxalic acid and HCN appears to be associated with enhanced elemental release from granite, in particular of Fe. This suggests that extensive microbial colonization of the granite surfaces could play a crucial role in the initial soil formation in previously glaciated mountain areas.Glaciers in alpine regions are highly sensitive to changes in climatic conditions (29). Increasing global atmospheric temperatures over the last decades have resulted in the recession of alpine glaciers (18). Forefields of temperate alpine glaciers provide unique opportunities to study initial soil formation as well as microbial and plant succession along the chronosequences (12, 26, 34, 36). The forefields close to the glacier terminus are initially vegetation free and consist mainly of rock material with high fractions of silt-sized grains with low C and N content and small amounts of available nutrients (14). Mineral weathering is a key process in the formation of soils (1, 26), and the crucial importance of microbially promoted mineral weathering for nutrient acquisition is increasingly recognized (2, 4, 39, 46). Recently exposed rock surfaces can be considered primary ecosystems where only a few microbes that are adapted due to their mineral-weathering abilities can grow (17). Some cations of rock-forming minerals are essential for proper cell functions. However, our understanding of geochemically significant microbes in forefields of temperate alpine glacier is still very limited but is crucial for increasing our knowledge of nutrient mobilization and the buildup of organic matter that is essential for the development of macroorganisms.The area of the Damma glacier in Central Switzerland is characterized by a relatively homogenous granitic rock basement and is used as field site of the interdisciplinary research project “Biosphere-Geosphere interactions: Linking climate change, weathering, soil formation and ecosystem evolution (BigLink)” (5). In the frame of this research project, we studied the functional roles of granite-colonizing microbes as biotic weathering agents in previously glaciated areas. So far, relatively little is known about microbe-granite interactions, especially regarding the release of trace elements. Several studies have examined the dissolution of specific granite-forming minerals in the presence of actively metabolizing bacteria or compounds that simulate metabolic activity (24, 30, 31, 37, 38, 44). There is a general agreement that microbially produced organic acids, siderophores, and extracellular polysaccharides can all promote dissolution of minerals. Previous dissolution experiments have mainly been performed with (i) commercially obtained minerals (23, 45), (ii) model microorganisms that were commercially obtained from culture collections (3, 35, 45), or (iii) laboratory strains, such as those of Bacillus subtilis (23) and Burkholderia fungorum (47). Most studies have focused on individual mineral specimens rather than on the mixture of minerals that are present in granite rock (47). Few studies observed mineral weathering of collected rock and bacteria isolated from volcanic areas covered with vegetation (30, 31). Moreover, there are no studies on microbial weathering for such immediately deglaciated environments combining functional and taxonomic investigations, probably due to the difficulties in obtaining heterotrophic bacterial isolates from granitic glacier forefields. In spite of this, a comprehensive culture collection containing approximately 500 bacterial strains, which were isolated from the glacier tongue of the Damma glacier, was established. Full-length 16S rRNA gene sequences of 120 isolates revealed that many isolates obtained from oligotrophic media were closely related to readily cultivable heterotrophic bacteria (e.g., Arthrobacter sp., Collimonas sp., Paenibacillus sp., and Pseudomonas sp.). These bacteria have been found to enhance mineral dissolution (39).Our aim was to characterize the impact of microorganisms on granite weathering. We performed laboratory dissolution experiments with sterile crushed granite rock material, 12 bacterial strains, and 1 algal strain. To investigate the potential weathering abilities of these isolates, granite dissolution experiments were performed abiotically with model agents, such as HCl for proton-promoted weathering or oxalate and citrate and KCN for ligand-promoted weathering.     

TaqMan Real-Time PCR Assays To Assess Arbuscular Mycorrhizal Responses to Field Manipulation of Grassland Biodiversity: Effects of Soil Characteristics,Plant Species Richness,and Functional Traits

Large-scale (temporal and/or spatial) molecular investigations of the diversity and distribution of arbuscular mycorrhizal fungi (AMF) require considerable sampling efforts and high-throughput analysis. To facilitate such efforts, we have developed a TaqMan real-time PCR assay to detect and identify AMF in environmental samples. First, we screened the diversity in clone libraries, generated by nested PCR, of the nuclear ribosomal DNA internal transcribed spacer (ITS) of AMF in environmental samples. We then generated probes and forward primers based on the detected sequences, enabling AMF sequence type-specific detection in TaqMan multiplex real-time PCR assays. In comparisons to conventional clone library screening and Sanger sequencing, the TaqMan assay approach provided similar accuracy but higher sensitivity with cost and time savings. The TaqMan assays were applied to analyze the AMF community composition within plots of a large-scale plant biodiversity manipulation experiment, the Jena Experiment, primarily designed to investigate the interactive effects of plant biodiversity on element cycling and trophic interactions. The results show that environmental variables hierarchically shape AMF communities and that the sequence type spectrum is strongly affected by previous land use and disturbance, which appears to favor disturbance-tolerant members of the genus Glomus. The AMF species richness of disturbance-associated communities can be largely explained by richness of plant species and plant functional groups, while plant productivity and soil parameters appear to have only weak effects on the AMF community.Arbuscular mycorrhizae are mutualistic associations between roots of plants and fungi that have been present for more than 400 million years (54). Approximately 80% of examined land plants (71), and almost all fungi of the phylum Glomeromycota (60), are capable of forming such associations. The main benefit of this relationship for plants is that it facilitates their acquisition of nutrients (especially P and N), while the fungus receives photoassimilates (7, 62). About 200 Glomeromycota species have been described to date, based on spore morphology (http://www.lrz-muenchen.de/∼schuessler/amphylo/amphylogeny.html), but there is increasing molecular evidence of significantly higher diversity in arbuscular mycorrhizal fungi (AMF) (10, 72).Diverse AMF communities have been detected in a wide range of plant communities (inter alia grasslands, boreal forests, and tropical communities; for an overview, see reference 48). Hence, AMF have been considered to be tolerant of wide ranges of ecological conditions and capable of associating with diverse plant partners. Identifying the factors regulating their community assemblages is challenging, but AMF community composition has been shown to be influenced by plant species diversity (e.g., see references 10, 22, and 33), and conversely, significant effects of AMF species and communities on the diversity and productivity of plant communities have been described (25, 68). Soil physicochemical parameters like phosphorus, nitrogen, and carbon availability (e.g., see references 4, 9, and 31); pH (17); moisture content (53); and disturbance (30) also reportedly influence AMF distribution. Hence, there is some support for niche theory, which presumes that two species of the same trophic level cannot coexist in a limited system and, if two species are present in such circumstances, one should become extinct (21). As a corollary, two cooccurring species must occupy niches that differ in some dimensions, e.g., plant hosts and/or soil properties (28). However, there are also indications that neutral ecological processes, as well as niche-defining parameters, may influence AMF diversity and community composition (17, 39). In contrast to niche theory, neutral theory (27) postulates that all individuals of every species at a given trophic level in a food web have ecological equivalence, and thus, all species within trophically defined communities can be regarded as open nonequilibrium assemblages that are solely shaped by dispersal and distinctions in spatiotemporal dimensions. According to the work of Hubbell (27), neutrality is defined at the level of individual organisms with identical probabilities of birth, death, migration, and speciation and not at the species level. In order to explore AMF communities more thoroughly and to test competing hypotheses, such as those raised by the niche and neutral theories, robust methods for high-throughput analyses of the communities are required.Recent investigations of variables that affect the structure of AMF communities have considered relationships between niche-defining dimensions, such as soil types (39) and pH gradients (17), and spatial variations in AMF community structure but not the role of plant diversity or functional traits of host plants. There have been several plant diversity manipulation experiments designed for coanalyzing multiple sets of ecological variables (e.g., the BIODEPTH and Cedar Creek projects) that would have been ideal for detailed examinations of effects of ecological variables on AMF, but previously reported AMF analyses in these experiments have been limited to counts of spores in a single study (11). However, not all AMF species regularly sporulate, and when present, spores poorly reflect AMF diversity (69), since active AMF occur as mycelia in roots and soils (e.g., see references 12 and 26). PCR-based molecular techniques enable much more rigorous characterization of AMF communities in these compartments (e.g., see references 26, 36, and 72), but assessments of broad spatial (42) and/or temporal (52) variations in AMF communities require analysis of large numbers of samples, which is not feasible using conventional PCR amplification followed by cloning and sequencing. This challenge can be potentially met by real-time PCR-based approaches, in which the AMF sequence types present in compartments of interest are first identified and then sequence type-specific probes are used for large-scale screening in real-time PCR TaqMan assays.In the study presented here, we explored AMF diversity in plots used in the Jena Experiment, a grassland plant diversity manipulation of 60 plant species representing four functional groups in 81 plots of 400 m² (56). The overall AMF diversity and community structure were first assessed by PCR amplification, cloning, and sequencing (55) of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences in soil samples from 23 representative plots. Using the acquired data, we then developed sequence type-specific probes, which were applied in high-throughput real-time PCR TaqMan assays of samples from all 81 experimental plots, and the effects of 15 plant and soil variables on the AMF community assemblage were investigated.     

Technical note: PCR analysis of minimum target amount of ancient DNA

The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low‐volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and β‐actin gene specific fragments were amplified via low‐volume PCR in a total reaction volume of 1 μl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low‐volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Protein dynamics detected in a membrane-embedded potassium channel using two-dimensional solid-state NMR spectroscopy

We report longitudinal ¹⁵N relaxation rates derived from two-dimensional (¹⁵N, ¹³C) chemical shift correlation experiments obtained under magic angle spinning for the potassium channel KcsA-Kv1.3 reconstituted in multilamellar vesicles. Thus, we demonstrate that solid-state NMR can be used to probe residue-specific backbone dynamics in a membrane-embedded protein. Enhanced backbone mobility was detected for two glycine residues within the selectivity filter that are highly conserved in potassium channels and that are of core relevance to the filter structure and ion selectivity.     

Characterization of two different acyl carrier proteins in complex I from Yarrowia lipolytica

Acyl carrier proteins of mitochondria (ACPMs) are small (∼ 10 kDa) acidic proteins that are homologous to the corresponding central components of prokaryotic fatty acid synthase complexes. Genomic deletions of the two genes ACPM1 and ACPM2 in the strictly aerobic yeast Yarrowia lipolytica resulted in strains that were not viable or retained only trace amounts of assembled mitochondrial complex I, respectively. This suggested different functions for the two proteins that despite high similarity could not be complemented by the respective other homolog still expressed in the deletion strains. Remarkably, the same phenotypes were observed if just the conserved serine carrying the phosphopantethein moiety was exchanged with alanine. Although this suggested a functional link to the lipid metabolism of mitochondria, no changes in the lipid composition of the organelles were found. Proteomic analysis revealed that both ACPMs were tightly bound to purified mitochondrial complex I. Western blot analysis revealed that the affinity tagged ACPM1 and ACPM2 proteins were exclusively detectable in mitochondrial membranes but not in the mitochondrial matrix as reported for other organisms. Hence we conclude that the ACPMs can serve all their possible functions in mitochondrial lipid metabolism and complex I assembly and stabilization as subunits bound to complex I.     

A Blind Spot in Confocal Reflection Microscopy: The Dependence of Fiber Brightness on Fiber Orientation in Imaging Biopolymer Networks

We investigate the dependence of fiber brightness on three-dimensional fiber orientation when imaging biopolymer networks with confocal reflection microscopy (CRM) and confocal fluorescence microscopy (CFM). We compare image data of fluorescently labeled type I collagen networks concurrently acquired using each imaging modality. For CRM, fiber brightness decreases for more vertically oriented fibers, leaving fibers above ∼50° from the imaging plane entirely undetected. As a result, the three-dimensional network structure appears aligned with the imaging plane. In contrast, CFM data exhibit little variation of fiber brightness with fiber angle, thus revealing an isotropic collagen network. Consequently, we find that CFM detects almost twice as many fibers as are visible with CRM, thereby yielding more complete structural information for three-dimensional fiber networks. We offer a simple explanation that predicts the detected fiber brightness as a function of fiber orientation in CRM.     

Residue-specific side-chain packing determines the backbone dynamics of transmembrane model helices

The transmembrane domains (TMDs) of membrane-fusogenic proteins contain an overabundance of β-branched residues. In a previous effort to systematically study the relation among valine content, fusogenicity, and helix dynamics, we developed model TMDs that we termed LV-peptides. The content and position of valine in LV-peptides determine their fusogenicity and backbone dynamics, as shown experimentally. Here, we analyze their conformational dynamics and the underlying molecular forces using molecular-dynamics simulations. Our study reveals that backbone dynamics is correlated with the efficiency of side-chain to side-chain van der Waals packing between consecutive turns of the helix. Leu side chains rapidly interconvert between two rotameric states, thus favoring contacts to its i±3 and i±4 neighbors. Stereochemical restraints acting on valine side chains in the α-helix force both β-substituents into an orientation where i,i±3 interactions are less favorable than i,i±4 interactions, thus inducing a local packing deficiency at VV3 motifs. We provide a quantitative molecular model to explain the relationship among chain connectivity, side-chain mobility, and backbone flexibility. We expect that this mechanism also defines the backbone flexibility of natural TMDs.     

Investigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls

Background

Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission.

Methods

Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic). Additionally, presence of antibodies against Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier^® ELISA Cp. abortus serum verification kit.

Results

No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus were detected.

Conclusions

The results suggest that Cp. abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low. However, because Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.     

Spatial patterns of Bovine Corona Virus and Bovine Respiratory Syncytial Virus in the Swedish beef cattle population

Background  

Both bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) infections are currently wide-spread in the Swedish dairy cattle population. Surveys of antibody levels in bulk tank milk have shown very high nationwide prevalences of both BCV and BRSV, with large variations between regions. In the Swedish beef cattle population however, no investigations have yet been performed regarding the prevalence and geographical distribution of BCV and BRSV. A cross-sectional serological survey for BCV and BRSV was carried out in Swedish beef cattle to explore any geographical patterns of these infections.