枯草芽孢杆菌近缘种群鉴定方法研究进展

枯草芽孢杆菌近缘种群(简称枯草群)是一群表型相似的革兰氏阳性、产芽孢的杆菌,目前该群已包括10个有效发表种。该群细菌广泛应用于生物技术、工业和农业等领域,因此实现菌株的快速准确鉴别与鉴定,确保其使用的安全性和有效性是首要条件。本文在本研究室进行的枯草群鉴定研究基础上,总结提出了基于形态和生化特性测定的传统经典方法和以分子生物学技术为基础的基因序列分析技术、特异PCR技术,并在实践上进行了大量的验证,对指导该种群的准确鉴定与快速鉴别具有重要应用价值。     

Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance

PCR amplification of 16S rRNA gene by universal primers followed by restriction fragment length polymorphism analysis using RsaI, CfoI and HinfI endonucleases, distinctly differentiated closely related Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus from Bacillus subtilis sensu stricto. This simple, economical, rapid and reliable protocol could be an alternative to misleading phenotype-based grouping of these closely related species.     

Development of species-specific primers for identification of Biomphalaria arabica,the intermediate host of Schistosoma mansoni in Saudi Arabia

Schistosoma mansoni is mediated through the intermediate host Biomphalaria arabica which lives in Saudi Arabia. Molecular characterization and identification of this intermediate host are important for epidemiological studies of schistosomiasis. The present work aimed to determine the molecular variations among the populations of B. arabica found in Southern part of Saudi Arabia, and to develop species-specific primers for identification of these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Five populations of Saudi B. arabica snails were collected from freshwater bodies. Three populations were collected from Asser and two populations were collected from AL-Baha. Genomic DNA was extracted from snails and was amplified using five different RAPD–PCR primers. The banding patterns of amplified materials by primers P1 and P5 were identical in all populations. However, the rest primers displayed intra-specific differences among populations with variable degrees. Largest sizes of RAPD–PCR products were cloned into TA cloning vector as a preparatory step for DNA sequence analysis. After sequencing, similarity searches of obtained DNA sequences revealed that there are no similar sequences submitted to genebank data bases and its associated banks. The results obtained will be helpful in the development of simultaneous identification of B. arabica snails and diagnosis of S. mansoni infection within it in a single step by an implementation of multiplex PCR.     

Typing of four genetic loci discriminates among closely related species of New World Leishmania

All New World Leishmania species can cause cutaneous lesions, while only Leishmania (Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.     

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

Background and Aims

Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera.

Methods

In order to isolate highly repetitive sequences, a c₀t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH).

Key Results

Characterization of the c₀t-1 library resulted in the detection of repetitive sequences including the (GA)_n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species.

Conclusions

The repeated sequences isolated from D. moschatum c₀t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.     

A FAFLP system for the improved identification of plant-pathogenic and plant-associated species of the genus Pantoea

The majority of Pantoea species are either plant-pathogenic or plant-associated and cause a wide variety of symptoms on a range of hosts. Identification of Pantoea species is difficult due to minor differences in phenotypic characteristics between them and related Enterobacteriaceae. Fluorescent amplified fragment length polymorphism (FAFLP) analysis was investigated for use as a rapid, molecular-based identification technique to the species level of the genus Pantoea. Following analysis of the band patterns generated by FAFLP, seven distinct clusters were observed, one for each validly published species of the genus. FAFLP has proven to be a rapid, reproducible identification technique for all species of the genus Pantoea.     

DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system

Recently, a new flow cytometric technology to detect multiple DNA target sequences in a single microtiter well plate was developed [multianalyte profiling (MAP) System, Luminex Corp., Austin, TX]. DNA probes, directed to the internal transcribed spacer 2 region of ribosomal DNA, were therefore designed to detect and differentiate PCR amplicons from six medically important Candida species using this system. Each probe was covalently linked to one of 100 available microsphere (bead) sets. Biotinylated PCR amplicons were then hybridized to the complementary probe on each bead set. Bound amplicons were detected fluorometrically using a streptavidin-linked reporter dye, R-phycoerythrin. Specific hybridization was noted for all six Candida species probes (mean sample-to-background ratio+/-standard error: Candida albicans, 58.7+/-1.2; Candida tropicalis, 53.2+/-3.8; Candida glabrata, 46.9+/-2.1; Candida parapsilosis, 59.9+/-1.6; Candida krusei, 54.7+/-3.7 vs. 0.9+/-0.03 for all heterologous Candida species DNA targets and vs. 1.0+/-0.1 for samples containing water instead of DNA; P < 0.001). The limit of test sensitivity was 0.5 pg of DNA. A sample could be processed and analyzed within 1 h post-PCR amplification. Therefore, the multianalyte profiling system was rapid, sensitive and specific for the detection and differentiation of the most medically important species of Candida.     

DNA probes for the identification of microorganisms

Summary The detection and identification of microorganisms is being carried out increasingly using DNA. Each organism has a unique DNA sequence which can be used to distinguish closely related organisms. Using PCR amplification and sequencing of ribosomal RNA genes we have developed DNA probes for a number of pathogenic bacteria and fungi. The development of DNA assays based on PCR has resulted in new questions which must be addressed including process carry-over contamination and inhibition of the PCR amplification reaction once the problems associated with the implementation of DNA assays are ironed out.     

华南地区3种具有不同入侵性的近缘植物对低温胁迫的敏感性

以五爪金龙(Ipomoea cairica (L.) Sweet,重度入侵植物)、裂叶牵牛(Ipomoea nil (L.) Choisy,轻度/非入侵植物)和三裂叶薯(Ipomoea triloba L.,非入侵植物)3种起源于热带美洲、且在华南地区具有不同入侵性的番薯属藤本植物作为研究对象,通过比较它们在低温胁迫下的生理响应探究3种植物对低温的敏感性与它们入侵性之间的关系。通过测定在不同温度(28、15、10 ℃)处理下植物的生物量、活性氧、渗透调节物质、根系活力、光合特征等生理指标发现,五爪金龙、裂叶牵牛及三裂叶薯均通过增加光合系统Ⅱ的热耗散、积累渗透调节物质以及增强根系活力来应对低温环境,但15 ℃的温度条件已经对3种植物形成较为强烈的胁迫作用,表现为H₂O₂和丙二醛的积累、光合系统Ⅱ受损、根部细胞死亡以及生物量、根长的极显著下降(P<0.01),证明3种植物对低温胁迫均具有较高的敏感性。综合比较3种植物各生理指标的响应幅度发现,它们对低温的耐受性表现为:五爪金龙 > 裂叶牵牛 > 三裂叶薯,这与它们在华南地区的入侵危害程度一致,暗示低温敏感性的差异可能是其入侵性差异的重要原因。结果表明,低温敏感性是影响外来植物入侵性和入侵区域的重要因素,五爪金龙较高的低温敏感性是限制其在华南以外地区形成入侵危害的重要原因。     

Rapid identification of nitrogen-fixing and legume-nodulating Burkholderia species based on PCR 16S rRNA species-specific oligonucleotides

Several novel N₂-fixing Burkholderia species associated with plants, including legume-nodulating species, have recently been discovered. Presently, considerable interest exists in studying the diazotrophic Burkholderia species, both for their ecology and their great potential for agro-biotechnological applications. However, the available methods used in the identification of these Burkholderia species are time-consuming and expensive. In this study, PCR species-specific primers based on the 16S rRNA gene were designed, which allowed rapid, easy, and correct identification of most known N₂-fixing Burkholderia. With this approach, type and reference strains of Burkholderia kururiensis, B. unamae, B. xenovorans, B. tropica, and B. silvatlantica, as well as the legume-nodulating B. phymatum, B. tuberum, B. mimosarum, and B. nodosa, were unambiguously identified. In addition, the PCR species-specific primers allowed the diversity of the diazotrophic Burkholderia associated with field-grown tomato and sorghum plants to be determined. B. tropica and B. xenovorans were the predominant species found in association with tomato, but the occurrence of B. tropica with sorghum plants was practically exclusive. The efficiency of the species-specific primers was validated with the detection of B. tropica and B. xenovorans from DNA directly recovered from tomato rhizosphere soil samples. Additionally, using PCR species-specific primers, all of the legume-nodulating Burkholderia were correctly identified, even from single nodules collected from inoculated common bean plants. These primers could contribute to rapid identification of the diazotrophic and nodulating Burkholderia species associated with important crop plants and legumes, as well as revealing their environmental distribution.     

Optimization of melting analysis with TaqMan probes for detection of KRAS,NRAS, and BRAF mutations

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔT_m (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5–3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity—all of the qualities required for clinical genotyping.     

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.     

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

Description and DNA barcoding of Crematogaster fraxatrix Forel, 1911 and two new closely related species from Cambodia and Indonesia (Hymenoptera,Formicidae)

Crematogaster fraxatrix Forel, 1911 and two new species, C. chhangi sp. n. and C. simboloni sp. n., are described from Cambodia and Indonesia, respectively. DNA sequences were generated for C. fraxarix and the two newly described species using 3 amplications of two regions of the mitochondrial gene COI with a total of 1129 bp. The mean interspecific divergences are 9.4% and 23.5% for C. fraxatrix vs. C. chhangi, C. simboloni, respectively. DNA sequences reveal that C. simboloni is found to be genetically distinct from the other two species, but C. chhangi is not distinct from C. fraxatrix.     

Mycorrhizal associations and reproductive isolation in three closely related Orchis species

Background and Aims

The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.

Methods

We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.

Results

Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.

Conclusions

Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.     

Mitochondrial phylogenomics and genetic relationships of closely related pine moth (Lasiocampidae: Dendrolimus) species in China,using whole mitochondrial genomes

Background

Pine moths (Lepidoptera; Bombycoidea; Lasiocampidae: Dendrolimus spp.) are among the most serious insect pests of forests, especially in southern China. Although COI barcodes (a standardized portion of the mitochondrial cytochrome c oxidase subunit I gene) can distinguish some members of this genus, the evolutionary relationships of the three morphospecies Dendrolimus punctatus, D. tabulaeformis and D. spectabilis have remained largely unresolved. We sequenced whole mitochondrial genomes of eight specimens, including D. punctatuswenshanensis. This is an unambiguous subspecies of D. punctatus, and was used as a reference for inferring the relationships of the other two morphospecies of the D. punctatus complex. We constructed phylogenetic trees from this data, including twelve published mitochondrial genomes of other Bombycoidea species, and examined the relationships of the Dendrolimus taxa using these trees and the genomic features of the mitochondrial genome.

Results

The eight fully sequenced mitochondrial genomes from the three morphospecies displayed similar genome structures as other Bombycoidea species in terms of gene content, base composition, level of overall AT-bias and codon usage. However, the Dendrolimus genomes possess a unique feature in the large ribosomal 16S RNA subunits (rrnL), which are more than 60 bp longer than other members of the superfamily and have a higher AC proportion. The eight mitochondrial genomes of Dendrolimus were highly conservative in many aspects, for example with identical stop codons and overlapping regions. But there were many differences in start codons, intergenic spacers, and numbers of mismatched base pairs of tRNA (transfer RNA genes).Our results, based on phylogenetic trees, genetic distances, species delimitation and genomic features (such as intergenic spacers) of the mitochondrial genome, indicated that D. tabulaeformis is as close to D. punctatus as is D. punctatus wenshanensis, whereas D. spectabilis evolved independently from D. tabulaeformis and D. punctatus. Whole mitochondrial DNA phylogenies showed that D. spectabilis formed a well-supported monophyletic clade, with a clear species boundary separating it from the other congeners examined here. However, D. tabulaeformis often clustered with D. punctatus and with the subspecies D. punctatus wenshanensis. Genetic distance analyses showed that the distance between D. tabulaeformis and D. punctatus is generally less than the intraspecific distance of D. punctatus and its subspecies D. punctatus wenshanensis. In the species delimitation analysis of Poisson Tree Processes (PTP), D. tabulaeformis, D. punctatus and D. punctatus wenshanensis clustered into a putative species separated from D. spectabilis. In comparison with D. spectabilis, D. tabulaeformis and D. punctatus also exhibit a similar structure in intergenic spacer characterization. These different types of evidence suggest that D. tabulaeformis is very close to D. punctatus and its subspecies D. punctatus wenshanensis, and is likely to be another subspecies of D. punctatus.

Conclusions

Whole mitochondrial genomes possess relatively rich genetic information compared with the traditional use of single or multiple genes for phylogenetic purposes. They can be used to better infer phylogenetic relationships and degrees of relatedness of taxonomic groups, at least from the aspect of maternal lineage: caution should be taken due to the maternal-only inheritance of this genome. Our results indicate that D. spectabilis is an independent lineage, while D. tabulaeformis shows an extremely close relationship to D. punctatus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1566-5) contains supplementary material, which is available to authorized users.     

Towards large-scale FAME-based bacterial species identification using machine learning techniques

In the last decade, bacterial taxonomy witnessed a huge expansion. The swift pace of bacterial species (re-)definitions has a serious impact on the accuracy and completeness of first-line identification methods. Consequently, back-end identification libraries need to be synchronized with the List of Prokaryotic names with Standing in Nomenclature. In this study, we focus on bacterial fatty acid methyl ester (FAME) profiling as a broadly used first-line identification method. From the BAME@LMG database, we have selected FAME profiles of individual strains belonging to the genera Bacillus, Paenibacillus and Pseudomonas. Only those profiles resulting from standard growth conditions have been retained. The corresponding data set covers 74, 44 and 95 validly published bacterial species, respectively, represented by 961, 378 and 1673 standard FAME profiles. Through the application of machine learning techniques in a supervised strategy, different computational models have been built for genus and species identification. Three techniques have been considered: artificial neural networks, random forests and support vector machines. Nearly perfect identification has been achieved at genus level. Notwithstanding the known limited discriminative power of FAME analysis for species identification, the computational models have resulted in good species identification results for the three genera. For Bacillus, Paenibacillus and Pseudomonas, random forests have resulted in sensitivity values, respectively, 0.847, 0.901 and 0.708. The random forests models outperform those of the other machine learning techniques. Moreover, our machine learning approach also outperformed the Sherlock MIS (MIDI Inc., Newark, DE, USA). These results show that machine learning proves very useful for FAME-based bacterial species identification. Besides good bacterial identification at species level, speed and ease of taxonomic synchronization are major advantages of this computational species identification strategy.     

New taxonomic markers for identification of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> and discrimination between closely related species

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from different host plants nodules originating from different regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identification of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to differentiate and define relationships within a group of R. leguminosarum strains.     

The use of arbitrarily primed PCR (AP-PCR) to develop taxa specific DNA probes of known sequence

Abstract A rapid polymerase chain reaction (PCR) method to obtain taxa-specific DNA probes has been developed. The oligonucleotide probes derived from the sequences of species-specific (or other taxa) random amplified polymorphic DNA (RAPD) fragments. The methodology was applied to design probes for the halophilic archael species Haloferax mediterranei . With this technique, DNA probes of known sequence can be generated easily and without any previous knowledge about the properties of the microorganisms.