固氮类芽孢杆菌YUPP-5中环糊精糖基转移酶基因的克隆与功能分析

从魔芋根际分离的固氮类芽孢杆菌Paenibacillus azotofixans YUPP-5对多种β-1,4糖苷键连接的多糖具有水解作用。通过构建该菌的fosmid文库,克隆到2 157 bp的基因片段,编码环糊精糖基转移酶。大肠杆菌中表达此酶,能降解葡甘聚糖、羧甲基纤维素钠盐、几丁质、木聚糖等多种β-1,4糖苷键连接的多糖,同时该酶还能以葡甘聚糖为底物生成β-1,4糖苷键连接的环糊精,而文献报道这种酶仅能利用α-1,4糖苷键连接的淀粉为底物生成环糊精;并展示了环糊精糖基转移酶的一些新功能。     

Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere

Abstract: A molecular method for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere was developed. The system consisted of polymerase chain reaction (PCR) amplification of part of the variable V1 to V4 regions of the 16S ribosomal RNA gene, followed by hybridization with a specific oligonucleotide probe homologous to part of the intervening region. In vitro specificity tests showed that the detection system worked specifically for P. azotofixans strains, and did not detect other Paenibacillus species or species of other bacterial genera. Vegetative cells of a rifampicin resistant P. azotofixans derivative were trackable in Flevo silt loam (FSL) soil in 24 h experiments using both selective plating and most probable number (MPN)-PCR combined with probing, and plate counts parallelled MPN-PCR estimations of numbers of specific targets. MPN-PCR allowed for the detection of down to 10² introduced cells per g of dry soil. Introduced P. azotofixans spores did not form colonies on selective plates, but were detectable via PCR. The P. azotofixans populations introduced into the silt loam soil suffered a slow decline of the detectable plate count over a period of 14 days. MPN-PCR revealed a similar decline of the number of specific DNA targets. Greater numbers of targets were found in wheat rhizosphere from Flevo silt loam soil, and these numbers persisted throughout the experiment. Soil drying resulted in enhanced persistence of the target sequences, whereas in a constantly moist soil the numbers of target sequences declined. Rewetting of dried soil resulted in declining target sequence numbers. The MPN-PCR detection method is adequate to assess the impact of stress conditions affecting P. azotofixans in FSL and probably other soils, since it abolishes the need for culturing or specific markers and is direct and unambiguous due to its high specificity.     

胶质类芽胞杆菌PCR快速检测方法

摘要:【目的】胶质类芽胞杆菌(Paenibacillus mucilaginosus) 是微生物肥料广泛应用的功能菌种之一,筛选并鉴定其特异性引物,建立该菌种快速检测方法,对微生物肥料产品检测和评价至关重要。【方法】本文筛选了胶质类芽胞杆菌基因间的一段非编码序列作为特异性引物(orf06701-F:5'-ATGGAGGAAACATGGGGTGA-3'/orf06701-R: 5'-TCAGGAATGAAGGCCCCCTT-3'),通过PCR 反应条件/ 体系的优化、特异性及灵敏度检测,建立了胶质类芽胞杆菌     

Short communication: Construction of a cloning vector for Paenibacillus polymyxa and P. azotofixans

A cloning vector that could replicate in Paenibacillus polymyxa, P. azotofixans and Bacillus subtilis was constructed using two Staphylococcus aureus plasmids. The recombinant plasmid confers chloramphenicol and erythromycin resistance and contains unique restriction sites for PvuII and BclI. The stability of pRJ45 was analysed.     

Evaluation of the Biolog automated microbial identification system

Biolog's identification system was used to identify 39 American Type Culture Collection reference taxa and 45 gram-negative isolates from water samples. Of the reference strains, 98% were identified to genus level and 76% to species level within 4 to 24 h. Identification of some authentic strains of Enterobacter, Klebsiella, and Serratia was unreliable. A total of 93% of the water isolates were identified.     

Evaluation of the Biolog automated microbial identification system.

Biolog's identification system was used to identify 39 American Type Culture Collection reference taxa and 45 gram-negative isolates from water samples. Of the reference strains, 98% were identified to genus level and 76% to species level within 4 to 24 h. Identification of some authentic strains of Enterobacter, Klebsiella, and Serratia was unreliable. A total of 93% of the water isolates were identified.     

Evaluation of the Biolog system for the identification of Bacillus anthracis

The potential of the Biolog system for the identification of Bacillus anthracis was evaluated. In-house generated databases allowed the correct identification of 19 of 20 isolates of B. anthracis within 24 h. Five strains of the closely related B. cereus/thuringiensis group were misidentified as B. anthracis. For this reason the test could only serve as a primary screen with further testing being required to confirm identity. In addition 20% of all the strains of bacilli examined during the study gave unreadable reaction profiles due to false-positive reactions.     

Evaluation of biodegradability by the reduction of Tetrazolium Violet in Biolog microplates

A novel biodegradation test using Biolog MT microplates was developed. The method was based on the reduction of Tetrazolium Violet during mineralization of organic substrates. Both a microbial mixed culture (activated sludge) and pure culture of a bacterium (Pseudomonas aeruginosa) were used as inocula to evaluate its applicability. The procedure was successfully demonstrated with the ozonated samples of p-nitrophenol. Compared with previous methods, the proposed method is fast and convenient to use in practice.     

Evaluation of the Biolog system for the identification of food and beverage yeasts

The inconvenience of conventional yeast identification methods has resulted in the development of rapid, commercial systems, mainly for clinical yeast species. The Biolog system (Biolog Inc., Hayward, CA, USA) is a new semi-automated, computer-linked technology for rapid identification of clinical and non-clinical yeasts. The system is based around a microtitre tray and includes assimilation and oxidation tests. This paper evaluates the Biolog system for the identification of 21 species (72 strains) of yeasts of food and wine origin. Species correctly identified included Saccharomyces cerevisiae , Debaryomyces hansenii , Yarrowia lipolytica , Kluyveromyces marxianus , Kloeckera apiculata , Dekkera bruxellensis and Schizosaccharomyces pombe. Zygosaccharomyces bailii and Zygosaccharomyces rouxii were identified correctly 50% of the time and Pichia membranaefaciens 20% of the time.     

Analysis of the bacterial strains using Biolog plates in the contaminated soil from Riyadh community

Routine manufacture, detonation and disposal of explosives in land and groundwater have resulted in complete pollution. Explosives are xenobiotic compounds, being toxic to biological systems, and their recalcitrance leads to persistence in the environment. The methods currently used for the remediation of explosive contaminated sites are expensive and can result in the formation of toxic products. The present study aimed to investigate the bacterial strains using the Biolog plates in the soil from the Riyadh community. The microbial strains were isolated using the spread plate technique and were identified using the Biolog method. In this study we have analyzed from bacterial families of soil samples, obtained from the different sites in 5 regions at Explosive Institute. Our results conclude that Biolog MicroPlates were developed for the rapid identification of bacterial isolates by sole-carbon source utilization and can be used for the identification of bacteria. Out of five communities, only four families of bacteria indicate that the microbial community lacks significant diversity in region one from the Riyadh community in Saudi Arabia. More studies are needed to be carried out in different regions to validate our results.     

尾矿区不同植被恢复模式下高效固氮菌的筛选及Biolog鉴定

为了从铁尾矿中获得高效土著固氮菌,采用选择性无氮培养基从不同恢复模式下铁尾矿土样中筛选高效固氮菌,并且通过乙炔还原法对分离出的48株固氮菌进行固氮酶活性测定。结果表明:筛选出两株具有较高固氮活性菌株Db1与Ec1,Biolog鉴定为固氮菌属(Azotobacter),固氮比活力分别为203.20 nmol·mg-1·h-1和307.23 nmol·mg-1·h-1。不同植被恢复模式、物种多样性、尾矿坡向、工程措施、恢复年限均对尾矿固氮菌株数量有影响。样地处于尾矿阳坡,或属于人造混交林,或植被恢复年限长,又或者实施六孔砖、坡岩造地等工程措施的尾矿植被恢复模式下筛得的固氮菌菌株数较多或固氮活力较高。     

Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization

Enhancement of the desulfurization activities of Paenibacillus strains 32O-W and 32O-Y were investigated using dibenzothiophene (DBT) and DBT sulfone (DBTS) as sources of sulphur in growth experiments. Strains 32O-W, 32O-Y and their co-culture (32O-W plus 32O-Y), and Vitreoscilla hemoglobin (VHb) expressing recombinant strain 32O-Yvgb and its co-culture with strain 32O-W were grown at varying concentrations (0·1–2 mmol l⁻¹) of DBT or DBTS for 96 h, and desulfurization measured by production of 2-hydroxybiphenyl (2-HBP) and disappearance of DBT or DBTS. Of the four cultures grown with DBT as sulphur source, the best growth occurred for the 32O-Yvgb plus 32O-W co-culture at 0·1 and 0·5 mmol l⁻¹ DBT. Although the presence of vgb provided no consistent advantage regarding growth on DBTS, strain 32O-W, as predicted by previous work, was shown to contain a partial 4S desulfurization pathway allowing it to metabolize this 4S pathway intermediate.     

Characterisation of Paenibacillus jamilae strains that produce exopolysaccharide during growth on and detoxification of olive mill wastewaters

A total of 10 bacterial strains were isolated from a compost of corn treated with olive mill wastewaters (OMW) and selected by their capacity to synthesize exopolysaccharides (EPS). Morphological, physiological, biochemical and nutritional tests were used for a phenotypic study. A numerical analysis showed that all strains were 90% similar to each other. A DNA–DNA hybridization assay confirmed that all the strains belonged to Paenibacillus jamilae species. All the characterized strains were able to produce EPS growing on OMW batch cultures. The strain which was able to produce the highest EPS yield was chosen to perform an assay for testing its putative detoxifying activity, and it showed to reduce more than half the toxic capacity of the OMW. The results presented in this study, indicated the possible perspectives for using these bacterial strains to produce EPS and contribute to the bioremediation of the waste waters that are produced in the olive oil elaboration process.     

Phenotypic and genetic diversity of Paenibacillus azotofixans strains isolated from the rhizoplane or rhizosphere soil of different grasses

Fifty-three strains identified as Paenibacillus azotofixans were isolated from the rhizoplane and rhizosphere of different grasses and from soil. To study the diversity within this species, four approaches were used: assessment of homology with a nifKDH probe in hybridization experiments; use of a selected 20-mer primer to produce RAPD profiles and of BOX-PCR to generate genomic fingerprintings; and phenotypic tests using the API50CH system. The API tests performed with the 53 P. azotofixans strains showed that all strains produced acid from 15 carbohydrates; using six other carbohydrates (sorbitol, dulcitol, tagatose, starch, glycogen and D -arabitol), the strains could be divided in five groups of related strains. All strains tested showed homology to Klebsiella pneumoniae nifKDH genes, resulting in 14 different hybridization patterns with this probe. Using RAPD-fingerprinting with one appropriate primer, 23 different amplification patterns were observed. The BOX-PCR approach confirmed the grouping suggested by the RAPD fingerprinting. A comparison of the 53 strains by similarity matrix analysis using the data obtained in all approaches resulted in a phenogram, grouping them into five broad groups at 74% similarity and into 27 subgroups at 94% similarity. At 100% similarity, 31 groups of strains could be formed, indicating a high degree of diversity among the strains tested. Overall, the diversity was independent from the origin of strains, since a variety of different groups was isolated from each plant studied. However, some clusters were dominant in wheat and sugarcane samples. The results indicated that the methods used here are sensitive indicators of diversity among the strains studied and can be applied as efficient and reliable means for further ecological and biogeographical studies.     

多粘类芽孢杆菌HY96-2产脂肽类抗真菌物质的研究

对一株已经商业化的可防治植物枯萎病的生防菌-多粘类芽孢杆菌HY96-2发酵液中抗真菌活性物质进行了分离纯化,采用酸化、正丁醇萃取、乙酸乙酯沉淀、硅胶柱层析、Sephadex LH-20柱层析、高效液相色谱(HPLC)等方法分离得到了一个抗真菌活性化合物6B,经NMR、MS、MS/MS等光谱学方法鉴定其为Fusaricidin A。琼脂扩散法抑菌试验结果表明,6B对西瓜枯萎病菌、水稻纹枯病菌、灰霉病菌等15种植物病原真菌的最小抑菌浓度为12.5～50μg/mL。盆栽实验结果表明,6B对黄瓜灰霉病具有明显的防治效果,当6B的浓度达到250μg/mL时,防治效果高达95%。     

Influence of growth conditions on the production of extracellular proteolytic enzymes in Paenibacillus peoriae NRRL BD-62 and Paenibacillus polymyxa SCE2

AIMS: To analyse the extracellular protease profile of two Paenibacillus species, Paenibacillus peoriae and Paenibacillus polymyxa, as well as how different growth media influenced its expression. METHODS AND RESULTS: Both bacteria were cultured in five media [Luria-Bertani broth, glucose broth, thiamine/biotin/nitrogen broth (TBN), trypticase soy broth and a defined medium] for 48 h at 32 degrees C. Our results showed a heterogeneous protease secretion pattern whose expression was dependent on medium composition. However, TBN induced the most quantitative and qualitative protease production on both Paenibacillus. The proteases were detected in neutral-alkaline pH range, being totally inhibited by 1,10-phenanthroline, a zinc-metalloprotease inhibitor. We also analysed the protease expression during the growth and, at least to P. peoriae, the most elevated protease activity was measured at 96 h, in which the highest number of spores and a low concentration of viable cells were observed. CONCLUSIONS: The results presented add P. peoriae and P. polymyxa to the list of neutral-alkaline extracellular protease producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Paenibacillus species are ubiquitous in nature, are capable to form resistant spores and to produce several hydrolytic enzymes, including proteases. However, only few data concerning the production of these enzymes are available. Proteases produced by Paenibacillus strains may represent new sources for biotechnological use.     

DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions

Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108 bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108 bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.     

Identification of Acinetobacter isolates using the Biolog identification system

The Biolog system was assessed for its ability to identify genospecies of Acinetobacter from a wastewater treatment plant. A success rate of 83% was achieved in identifying to genus level, but different genospecies identifications were obtained, with the possible exception of genospecies 7, when the results were compared with other published phenotypic identification schemes.