Centrosema is a promiscuous legume nodulated by several new putative species and symbiovars of Bradyrhizobium in various American countries

Centrosema is an American indigenous legume that can be used in agroecosystems for recovery of acidic and degraded soils. In this study, a Centrosema-nodulating rhizobial collection of strains isolated in a poor acid savanna soil from Venezuela was characterized, and the members of the collection were compared to other Centrosema strains from America. The analysis of the rrs gene showed that the strains nodulating Centrosema in American countries were closely related to different species of the genus Bradyrhizobium. However, the analysis of the atpD and recA genes, as well as the 16S–23S ITS region, showed that they formed several new phylogenetic lineages within this genus. The Venezuela strains formed three lineages that were divergent among themselves and with respect to those formed by Centrosema strains isolated in other countries, as well as to the currently described species and genospecies of Bradyrhizobium. In addition, the symbiotic genes nodC and nifH carried by Centrosema-nodulating strains were analyzed for the first time, and it was shown that they belonged to three new phylogenetic lineages within Bradyrhizobium. The nodC genes of the Centrosema strains were divergent among themselves and with respect to the genistearum and glycinearum symbiovars, indicating that Centrosema is a promiscuous legume. According to these results, the currently known Centrosema-nodulating strains represent several new putative species and symbiovars of the genus Bradyrhizobium.     

Optimization of Campylobacter growth conditions for further identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Growth conditions – including growth medium and incubation temperature – may influence the identification of Campylobacter by MALDI-TOF MS.     

The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense)

Genista versicolor is an endemic legume from Sierra Nevada National Park which constitutes one of the UNESCO-recognized Biosphere Reserves. In the present study, a collection of strains nodulating this legume was analysed in characteristic soils of this ecosystem. Most strains nodulating G. versicolor belonged to rrs group I within the genus Bradyrhizobium and only one strain, named GV137, belonged to rrs group II from which only a single species, B. retamae, has been described in Europe to date. Strain GV137, and some strains from rrs group I, belonged to putative new species of Bradyrhizobium, although most strains from group I belonged to B. canariense, according to the ITS fragment and atpD gene analysis. This result contrasted with those obtained in Genista tinctoria in Northeast Europe whose endosymbionts were identified as B. japonicum. The analysis of the symbiotic nodC and nifH genes carried by G. versicolor-nodulating strains showed that most of them belonged to symbiovar genistearum, as did those isolated from G. tinctoria. Nevertheless, strain GV137, belonging to rrs group II, formed a divergent lineage that constituted a novel symbiovar within the genus Bradyrhizobium for which the name sierranevadense is proposed. This finding showed that the Genisteae are not restrictive legumes only nodulated by symbiovar genistearum, since Genista is a promiscuous legume nodulated by at least two symbiovars of Bradyrhizobium, as occurs in Retama species.     

Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.     

Multilocus sequence analysis of the genus Bradyrhizobium

The use of multilocus sequence analysis (MLSA) for the taxonomy of Bradyrhizobium was assessed. We compared partial sequences for atpD, recA, gyrB, rpoB and dnaK for a set of reference strains representing named species and genospecies, and a number of new isolates from Lupinus albus, Arachis hypogaea and Ornithopus compressus from Spain. The phylogenies of the individual genes were compared with previous DNA–DNA hybridization results. High hybridization values were well reflected, but intermediary hybridization values were less clearly apparent. However, the phylogeny of a concatenated dataset of the five genes did reflect all values and thus is more informative of overall genome similarity. Our results indicate that only for the genes gyrB, rpoB and dnaK there is a small gap between the interspecies sequence similarities and the intraspecies similarity, and therefore cut-off levels for species delineation cannot be set, although high sequence similarity (>99%) does permit identification. In a few instances, a reference strain did not group as expected for one of the five genes tested. This may be a result of horizontal gene transfer and recombination events occasionally involving housekeeping genes. This observation indicates it is best to consider more than one gene for taxonomic inferences. The majority of the new isolates from the three host species was identified as Bradyrhizobium canariense. Four strains from L. albus from León, Spain, formed a separate group close to Bradyrhizobium japonicum.     

Typing of nitrogen-fixing Frankia strains by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry

Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was evaluated as a technique to characterize strains of the nitrogen-fixing actinomycete Frankia. MALDI-TOF MS reliably distinguished 37 isolates within the genus Frankia and assigned them to their respective host infection groups, i.e., the Alnus/Casuarina and the Elaeagnus host infection groups. The assignment of individual strains to sub-groups within the respective host infection groups was consistent with classification based on comparative sequence analysis of nifH gene fragments, confirming the usefulness of MALDI-TOF MS as a rapid and reliable tool for the characterization of Frankia strains.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

Vigna unguiculata is nodulated in Spain by endosymbionts of Genisteae legumes and by a new symbiovar (vignae) of the genus Bradyrhizobium

Vigna unguiculata was introduced into Europe from its distribution centre in Africa, and it is currently being cultivated in Mediterranean regions with adequate edapho-climatic conditions where the slow growing rhizobia nodulating this legume have not yet been studied. Previous studies based on rrs gene and ITS region analyses have shown that Bradyrhizobium yuanmingense and B. elkanii nodulated V. unguiculata in Africa, but these two species were not found in this study. Using the same phylogenetic markers it was shown that V. unguiculata, a legume from the tribe Phaseolae, was nodulated in Spain by two species of group I, B. cytisi and B. canariense, which are common endosymbionts of Genisteae in both Europe and Africa. These species have not been found to date in V. unguiculata nodules in its African distribution centres. All strains from Bradyrhizobium group I isolated in Spain belonged to the symbiovar genistearum, which is found at present only in Genisteae legumes in both Africa and Europe. V. unguiculata was also nodulated in Spain by a strain from Bradyrhizobium group II that belonged to a novel symbiovar (vignae). Some African V. unguiculata-nodulating strains also belonged to this proposed new symbiovar.     

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic “fingerprints” are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.     

Phylogenetic diversity of Rhizobium strains nodulating diverse legume species growing in Ethiopia

The taxonomic diversity of thirty-seven Rhizobium strains, isolated from nodules of leguminous trees and herbs growing in Ethiopia, was studied using multilocus sequence analyses (MLSA) of six core and two symbiosis-related genes. Phylogenetic analysis based on the 16S rRNA gene grouped them into five clusters related to nine Rhizobium reference species (99–100% sequence similarity). In addition, two test strains occupied their own independent branches on the phylogenetic tree (AC86a2 along with R. tibeticum; 99.1% similarity and AC100b along with R. multihospitium; 99.5% similarity). One strain from Milletia ferruginea was closely related (>99%) to the genus Shinella, further corroborating earlier findings that nitrogen-fixing bacteria are distributed among phylogenetically unrelated taxa. Sequence analyses of five housekeeping genes also separated the strains into five well-supported clusters, three of which grouped with previously studied Ethiopian common bean rhizobia. Three of the five clusters could potentially be described into new species. Based on the nifH genes, most of the test strains from crop legumes were closely related to several strains of Ethiopian common bean rhizobia and other symbionts of bean plants (R. etli and R. gallicum sv. phaseoli). The grouping of the test strains based on the symbiosis-related genes was not in agreement with the housekeeping genes, signifying differences in their evolutionary history. Our earlier studies revealing a large diversity of Mesorhizobium and Ensifer microsymbionts isolated from Ethiopian legumes, together with the results from the present analysis of Rhizobium strains, suggest that this region might be a potential hotspot for rhizobial biodiversity.     

MALDI-TOF mass spectrometry and PSD fragmentation as means for the analysis of condensed tannins in plant leaves and needles

MALDI-TOF mass spectrometry and 13C NMR spectroscopy were applied to unveil typical characteristics of condensed tannins of leaves and needles from willow (Salix alba), spruce (Picea abies) and beech (Fagus sylvatica) of three tree species that are ubiquitous in German forests and landscapes. For further evaluation, lime (Tilia cordata) was included. The 13C NMR spectroscopy confirmed the purity of the condensed tannin fractions and the efficiency of the procedure used for their extraction. While signals representative for procyanidin units are observable in all liquid-state 13C NMR spectra, resonance lines of prodelphinidin were only detected in those obtained from the condensed tannins of spruce needles and beech leaves. Typical signals in the chemical shift region between 70 and 90 ppm demonstrated the presence of stereoisomers (catechin/epicatechin; gallocatechin/ epigallocatechin). The MALDI-TOF mass spectra of the condensed tannins show signals of polymers of up to undecamers. Supporting the observations from the NMR spectroscopy, the mass spectra of the willow and lime leaf condensed tannins were identified as polymers with mainly procyanidin units, while the polymers of the spruce needle and beech leaves exhibit varying procyanidin/prodelphinidin ratios. Post source decay (PSD) fragmentation lead to a sequential loss of monomers and allowed a detailed characterization and sequencing of individual chains. In the case of the condensed tannins of lime this technique clearly excludes a pelargonidin terminal unit followed by a prodelphinidin unit, which would result in the same molecular masses as a polymer solely built up of procyanidin units.     

Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives

The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species-complexes of morphologically similar taxa. This study aimed to assess if strains of four closely-related plant pathogens, i.e., accurately Alternaria dauci (ten strains), Alternaria porri (six), Alternaria solani (ten), and Alternaria tomatophila (ten) could be identified using multilocus phylogenetic analysis and Matrix-Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) profiling of proteins. Phylogenetic analyses were performed on three loci, i.e., the internal transcribed spacer (ITS) region of rRNA, and the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major antigen (Alt a 1) genes. Phylogenetic trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades. However, none provided significant bootstrap support for all four species, which could only be achieved when results for the three loci were combined. MALDI-TOF-based dendrograms showed three major clusters. The first comprised all A. dauci strains, the second included five strains of A. porri and one of A. solani, and the third included all strains of A. tomatophila, as well as all but one strain of A. solani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely-related plant pathogens.     

基质辅助激光解吸电离飞行时间质谱技术作为常见益生菌菌种鉴定辅助工具的研究

目的评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术用于常见益生菌菌株鉴定及潜在益生菌菌株筛选的可行性。方法利用16S rDNA序列分析在方法学上对MALDI-TOF MS技术的鉴定能力进行研究;通过MALDI-TOF MS技术对现有保藏菌株的鉴定结果研究MALDI-TOF MS技术的鉴定准确性及优越性。结果 MALDI-TOF MS技术具备较16S rDNA序列分析更高的菌株鉴定能力;MALDI-TOF MS技术的鉴定结果准确、稳定。结论 MALDI-TOF MS技术可以作为准确、快速、廉价及可高通量操作的菌株鉴定方法应用于常见益生菌菌株的鉴定及潜在益生菌菌株的筛选。     

Identification of tick periviscerokinin, the first neurohormone of Ixodidae: single cell analysis by means of MALDI-TOF/TOF mass spectrometry

The first peptidergic neurohormone from the ticks Ixodes ricinus and Boophilus microplus has been identified by using a combination of immunocytochemistry and mass spectrometric analysis of single cells. The novel peptide (Ixori-PVK, PALIPFPRV-NH2) shows a high sequence homology with members of the insect periviscerokinin/CAP2b peptides that in insects are involved in the regulation of water balance. The function of this peptide in ticks is still unknown, but these pests consume large amounts of blood in a single blood meal which is a challenge for the regulation of diuretic processes. Thus, the novel peptide may be involved in one of the key physiological processes in ticks. High energy collision-induced dissociation was successfully used to distinguish between Leu/Ile ambiguities in single cell preparations. This is the first successful de novo sequencing of a peptide from single cell preparations of arthropods.     

Ribosomal protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phylogenety-based subspecies resolution of Bifidobacterium longum

The taxonomic positions of the subspecies of Bifidobacterium longum (B. longum subsp. longum, subsp. infantis, and subsp. suis) have been controversial. A current proposal is that the former two species “B. infantis” and “B. suis” be unified with B. longum and all three reclassified as three subspecies. To test this proposal, ribosomal protein profiling as observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied to the classification of 17 strains of B. longum, including three subspecies. Among 41 different kinds of ribosomal proteins selected as biomarkers whose masses were calculated from their amino acid sequences, 31-41 ribosomal proteins were observed in sample strains with the same masses as the references. The high matching rate indicates high conservation of ribosomal proteins within the sample strains, and therefore strongly supports the unification of the former species. However, the masses of some ribosomal proteins varied within species. The phylogenetic tree constructed from the profiles of ribosomal proteins matched the references, showing a clear cluster of the subsp. longum and the subsp. infantis strains. This result supports the proposal to reclassify B. longum into subsp. longum and subsp. infantis. The subsp. suis strains formed an individual sub-cluster within the infantis cluster. However, their ribosomal proteins have both characters of longum and infantis types. This result suggests that the taxonomic position of the subsp. suis should be reconsidered.     

Diagnostic accuracy of MALDI-TOF mass spectrometry for non-small cell lung cancer: a meta-analysis

Objective: To assess the overall accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying non-small cell lung cancer (NSCLC).

Methods: A comprehensive search of PubMed, EMBASE and CNKI databases as well as the reference lists from relevant articles was performed prior to July 2017. Two authors independently screened articles based on inclusion and exclusion criteria and assessed the quality of each study using the Quality Assessment of Diagnostic Accuracy Studies 2 (QADAS-2) tool. Meta-disc 1.4 and Stata12.0 software programs were used for the statistical analysis.

Results: Eleven eligible articles comprising 16 studies and representing 935 subjects were included in this meta-analysis. The pooled sensitivity and specificity were 0.84 (95% CI: 0.80–0.87) and 0.77 (95% CI: 0.74–0.80), respectively. The overall diagnostic performance as measured by the area under the curve (AUC) for the summary receiver-operating characteristic (SROC) curve was 0.9380.

Conclusions: MALDI-TOF MS has a high diagnostic accuracy for NSCLC.     

Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain

The genomic diversity of a collection of 103 indigenous rhizobia isolates from Lupinus mariae-josephae (Lmj), a recently described Lupinus species endemic to alkaline-limed soils from a restricted habitat in Eastern Spain, was investigated by molecular methods. Isolates were obtained from soils of four geographic locations in the Valencia province that harbored the known Lmj plant populations. Using an M13 RAPD fingerprinting technique, 19 distinct RAPD profiles were identified. Phylogenetic analysis based on 16S rDNA and the housekeeping genes glnII, recA and atpD showed a high diversity of native Bradyrhizobium strains that were able to establish symbiosis with Lmj. All the strains grouped in a clade unrelated to strains of the B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. The phylogenetic tree based on concatenated glnII, recA and atpD gene sequences grouped the Lmj isolates in six different operational taxonomic units (OTUs) at the 93% similarity level. These OTUs were not associated to any specific geographical location, and their observed divergence predicted the existence of different Bradyrhizobium genomic species. In contrast, phylogenetic analysis of symbiotic genes based on nodC and nodA gene sequences, defined only two distinct clusters among the Lmj strains. These two Lmj nod gene types were largely distinct from nod genes of bradyrhizobia nodulating other Old World lupine species. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host.     

Streamlining plant sample preparation: the use of high-throughput robotics to process echinacea samples for biomarker profiling by MALDI-TOF mass spectrometry.

Several species in the genus Echinacea are beneficial herbs popularly used for many ailments. The most popular Echinacea species for cultivation, wild collection, and herbal products include E. purpurea (L.) Moench, E. pallida (Nutt.) Nutt., and E. angustifolia (DC). Product adulteration is a key concern for the natural products industry, where botanical misidentification and introduction of other botanical and nonbotanical contaminants exist throughout the formulation and production process. Therefore, rapid and cost-effective methods that can be used to monitor these materials for complex product purity and consistency are of benefit to consumers and producers. The objective of this continuing research was to develop automated, high-throughput processing methods that, teamed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, differentiate Echinacea species by their mass profiles. Small molecules, peptide, and proteins from aerial parts (leaf/stem/flowers), seeds, and roots from E. purpurea and E. angustifolia; seeds and roots from E. pallida; and off-the-shelf Echinacea supplements were extracted and analyzed by MS using methods developed on the ProPrep liquid handling system (Genomic Solutions). Analysis of these samples highlighted key MS signal patterns from both small molecules and proteins that characterized the individual Echinacea materials analyzed. Based on analysis of pure Echinacea samples, off-the-shelf products containing Echinacea could then be evaluated in a streamlined process. Corresponding analysis of dietary supplements was used to monitor for product composition, including Echinacea species and plant materials used. These results highlight the potential for streamlined, automated approaches for agricultural species differentiation and botanical product evaluation.     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.