Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms

Bacillus strains were isolated from the rhizosphere of cereals in order to be used as natural biocontrol agents (BCAs). They were screened for antagonism in vitro against various test micro-organisms. The isolates showing antagonism were identified to species level. A combination of techniques was employed for the isolation of Bacillus species. Using the direct method, only one of the 25 isolates screened showed antagonistic properties. This strain (IFS-01) was identified by means of API test strips and the ATB Plus computer programme. It proved to be Bacillus subtilis and consequently has been designated as Bacillus subtilis IFS-01. This strain produced either a broad spectrum antimicrobial compound or several compounds with different activities. The fungi and Gram-positive bacteria were more sensitive to the antagonistic isolate than the Gram-negative bacteria. A Bacillus strain producing BCAs which can be used as biopesticides or organic preservatives has been isolated and identified.     

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.     

Use of single-point genome signature tags as a universal tagging method for microbial genome surveys

We developed single-point genome signature tags (SP-GSTs), a generally applicable, high-throughput sequencing-based method that targets specific genes to generate identifier tags from well-defined points in a genome. The technique yields identifier tags that can distinguish between closely related bacterial strains and allow for the identification of microbial community members. SP-GSTs are determined by three parameters: (i) the primer designed to recognize a conserved gene sequence, (ii) the anchoring enzyme recognition sequence, and (iii) the type IIS restriction enzyme which defines the tag length. We evaluated the SP-GST method in silico for bacterial identification using the genes rpoC, uvrB, and recA and the 16S rRNA gene. The best distinguishing tags were obtained with the restriction enzyme Csp6I upstream of the 16S rRNA gene, which discriminated all organisms in our data set to at least the genus level and most organisms to the species level. The method was successfully used to generate Csp6I-based tags upstream of the 16S rRNA gene and allowed us to discriminate between closely related strains of Bacillus cereus and Bacillus anthracis. This concept was further used successfully to identify the individual members of a defined microbial community.     

Use of Single-Point Genome Signature Tags as a Universal Tagging Method for Microbial Genome Surveys

We developed single-point genome signature tags (SP-GSTs), a generally applicable, high-throughput sequencing-based method that targets specific genes to generate identifier tags from well-defined points in a genome. The technique yields identifier tags that can distinguish between closely related bacterial strains and allow for the identification of microbial community members. SP-GSTs are determined by three parameters: (i) the primer designed to recognize a conserved gene sequence, (ii) the anchoring enzyme recognition sequence, and (iii) the type IIS restriction enzyme which defines the tag length. We evaluated the SP-GST method in silico for bacterial identification using the genes rpoC, uvrB, and recA and the 16S rRNA gene. The best distinguishing tags were obtained with the restriction enzyme Csp6I upstream of the 16S rRNA gene, which discriminated all organisms in our data set to at least the genus level and most organisms to the species level. The method was successfully used to generate Csp6I-based tags upstream of the 16S rRNA gene and allowed us to discriminate between closely related strains of Bacillus cereus and Bacillus anthracis. This concept was further used successfully to identify the individual members of a defined microbial community.     

小龙虾肠道产木聚糖酶细菌的分离与鉴定

【背景】小龙虾肠道微生物是小龙虾降解纤维素和半纤维素的主要驱动力。【目的】研究肠道内细菌的相对丰度,为揭示肠道微生物在小龙虾纤维素降解过程中的作用提供理论支撑。【方法】采用纯培养法从小龙虾肠道筛选产木聚糖酶细菌,并且对小龙虾肠道细菌进行16S高通量测序。【结果】形态学和16SrRNA基因分子鉴定表明,筛选到的4株产木聚糖酶细菌均属于芽孢杆菌科芽孢杆菌属;结合进一步的生理生化特征鉴定,结果为:菌株Z-3为枯草芽孢杆菌(Bacillus subtilis),菌株Z-4为贝莱斯芽孢杆菌(Bacillus velezensis),菌株Z-29为蜡状芽孢杆菌(Bacillus cereus),菌株Z-30为高地芽孢杆菌(Bacillus altitudinis);16S rRNA基因高通量测序结果表明:在属水平上,小龙虾肠道细菌主要是Candidatus Bacilloplasma、拟杆菌属、弧菌属、不动杆菌属、Dysgonomonas、Tyzzerella3、气单胞菌属和希瓦氏菌属细菌。【结论】小龙虾肠道内细菌资源丰富,且芽孢杆菌属细菌在木质纤维素降解过程中发挥一定功能。     

Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach

A new chemotaxonomic method is presented for the identification of eubacteria. This method is based on one-dimensinal gel electrophoresis of total RNA extracts from eubacteria. Only low molecular weight (<150 nucleotides) RNA, comprising 5S ribosomal and transfer RNA, was used for the identification. The high resolution of the electrophoresis, better than half a nucleotide, allowed construction of low molecular weight (LMW) RNA profiles that contained 10 to 20 bands per strain. LMW RNA profiles of a set of eubacterial reference strains showed on variation in dependence on culture conditions or physiological state of the cells. Computer-assisted data evaluation, including six molecular weight markers, enabled the calculation of relative nucleotide units (RNU) for every band. The resulting normalized band pattern allowed the identification of identical strains on different gels. The relative position of the single bands from the different groups of RNAs made an identification of bacterial strains to genus and often species level possible.Especially valuable for the identification were the large, class 2 tRNAs that showed certain variation among species of the same genus and varied considerably among different genera. RNA profiles can provide a rapid and inexpensive screening technique for the taxonomic classification of single bacterial strains. Potential fields of application for this technique might be bacterial taxonomy, biotechnology and ecology.     

Streptomyces pharetrae sp. nov., isolated from soil from the semi-arid Karoo region

An actinomycete, strain CZA14T, was isolated from Worcester in the Western Cape province of South Africa. Based on rapid genus identification, 16S-rDNA sequence similarities and chemotaxonomy, strain CZA14T was identified as a member of the genus Streptomyces. It exhibited weak antibiosis against Bacillus coagulans ATCC 7050T, Mycobacterium aurum A+ and Acinetobacter calcoaceticus C91. The results of physiological tests and analysis of the 16S-rDNA sequence allowed for the differentiation of strain CZA14T from other species of the genus Streptomyces. Strain CZA14T therefore represents a new species for which the name Streptomyces pharetrae is proposed, with the type strain CZA14T (= DSM 41856T = NRRL 24333T).     

Molecular weight variation among bacterial flagellins.

The molecular weights of flagellins from 29 bacterial strains from 11 genera were investigated using the technique of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weights range from 28,600 for Bacillus sphaericus to 63,200 for one strain of Escherichia coli. Variation in molecular weight was observed not only between genera but also between different species of a genus and different strains of a species.     

The scope and limitations of four methods for the rapid identification of Enterobacteriaceae in foods

Four methods for the identification of Enterobacteriaceae in cooked frozen meat have been appraised. Although these methods could be modified to improve efficiency they could be clearly classified into two categories: (i) those that allow identification to genus level and which often cannot discriminate between two genera without the help of additional reactions; (ii) those that allow identification to species level and which only occasionally require additional reactions to differentiate one species from another. A different evaluation criterion was followed for each of these categories. In the first, as methods lead to final identification by means of data furnished by the manufacturer or via a number code which does not indicate probability, reactions were compared with those obtained by classical methods. In the second group, every number code admits three possibilities and indicates a probable biotype for each, thus reducing the risk of faulty interpretation on the part of the operator while also compensating for deficiencies of certain reactions. Evaluation was therefore based on the percentage of correctly identified cases per species. Efficiencies of the main biochemical reactions are also discussed.     

The scope and limitations of four methods for the rapid identification of Enterobacteriaceae in foods

Four methods for the identification of Enterobacteriaceae in cooked frozen meat have been appraised. Although these methods could be modified to improve efficiency they could be clearly classified into two categories: (i) those that allow identification to genus level and which often cannot discriminate between two genera without the help of additional reactions; (ii) those that allow identification to species level and which only occasionally require additional reactions to differentiate one species from another. A different evaluation criterion was followed for each of these categories. In the first, as methods lead to final identification by means of data furnished by the manufacturer or via a number code which does not indicate probability, reactions were compared with those obtained by classical methods. In the second group, every number code admits three possibilities and indicates a probable biotype for each, thus reducing the risk of faulty interpretation on the part of the operator while also compensating for deficiencies of certain reactions. Evaluation was therefore based on the percentage of correctly identified cases per species. Efficiencies of the main biochemical reactions are also discussed.     

Development of a Simple and Rapid Method Based on Polymerase Chain Reaction–Based Restriction Fragment Length Polymorphism Analysis to Differentiate Helicobacter, Campylobacter, and Arcobacter Species

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of amplified DNA fragment of the 16S and 23S rRNA genes was performed on 35 Helicobacter, 24 Campylobacter, and 15 Arcobacter strains. PCR amplification generated a 1004-bp fragment of 16S rDNA and a 2.6-Kbp fragment of 23S rDNA from each strain. The amplicons were digested with DdeI and HpaII, respectively. For both assays, distinctive profiles were obtained for each genus. 23S rDNA PCR-RFLP analysis with HpaII enzyme identified Campylobacter and Helicobacter strains at the species level. Analysis of 16S rRNA gene with DdeI enzyme was not useful for the specific identification of Campylobacter and Arcobacter, although it discriminated among Helicobacter species. The PCR-RFLP technique allowed for the discrimination among these three related genus with only one restriction enzyme; therefore it can be a simple, rapid, and useful method for routine identification.     

姜科砂仁属植物DNA条形码序列的筛选

砂仁属（Amomum）隶属于姜科,全属约150种,我国有39种。该属多种植物可作药物或香料,但目前砂仁属的分类还不清楚,准确鉴定物种有很大难度。本研究利用DNA barcoding技术,对砂仁属50种121个个体的matK、rbcL-a、trnH-psbA序列及其不同组合进行比较,用Taxon DNA计算种间、种内bar-coding gap,运用相似法的BLASTn计算条码的正确鉴定率,筛选适合砂仁属的条码片段。结果显示：所有条码的barcoding gap均不存在;matK的正确鉴定率高于trnH-psbA和rbcL-a,联合片段的条码正确鉴定率高于单片段条码,三个片段联合条码的正确鉴定率最高。因此,推荐matK＋rbcL-a＋trnH-psbA作为砂仁属物种鉴定的候选条码。     

设计乳杆菌特异性引物并运用菌落PCR技术快速检出和鉴定四川泡菜中的乳杆菌

乳杆菌(Lactobacillus)是益生菌, 也是当前的研究热点之一。研究泡菜等样品中的乳杆菌需要快速的检出方法。根据已完成全基因组测序的14种乳杆菌的16S rDNA序列, 设计一对乳杆菌特异性引物。PCR检测结果表明该引物对乳杆菌和明串珠菌能扩增出800 bp的片段, 对表皮葡萄球菌、乳酸乳球菌和枯草芽胞杆菌却没有扩增条带, 具有一定的乳杆菌特异性。结合MRS乳杆菌半选择培养基和革兰氏染色, 运用菌落PCR技术, 可以快速高效地检出四川泡菜中的乳杆菌。再通过对PCR扩增片段测序, 可以将乳杆菌鉴定到种。从16份四川泡菜样品中检出了15株乳杆菌, 其中14株被鉴定为植物乳杆菌, 1株需进一步鉴定才能确定种。该方法可以检出乳杆菌新种。     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

Computer-assisted pattern recognition model for the identification of slowly growing mycobacteria including Mycobacterium tuberculosis.

We present a computerized pattern recognition model used to speciate mycobacteria based on their restriction fragment length polymorphism (RFLP) banding patterns. DNA fragment migration distances were normalized to minimize lane-to-lane variability of band location both within and among gels through the inclusion of two internal size standards in each sample. The computer model used a library of normalized RFLP patterns derived from samples of known origin to create a probability matrix which was then used to classify the RFLP patterns from samples of unknown origin. The probability matrix contained the proportion of bands that fell within defined migration distance windows for each species in the library of reference samples. These proportions were then used to compute the likelihood that the banding pattern of an unknown sample corresponded to that of each species represented in the probability matrix. As a test of this process, we developed an automated, computer-assisted model for the identification of Mycobacterium species based on their normalized RFLP banding patterns. The probability matrix contained values for the M. tuberculosis complex, M. avium, M. intracellulare, M. kansasii and M. gordonae species. Thirty-nine independent strains of known origin, not included in the probability matrix, were used to test the accuracy of the method in classifying unknowns: 37 of 39 (94.9%) were classified correctly. An additional set of 16 strains of known origin representing species not included in the model were tested to gauge the robustness of the probability matrix. Every sample was correctly identified as an outlier, i.e. a member of a species not included in the original matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computer-assisted method for identification of Bacillus species isolated from liquid antacids

One hundred nineteen species of Bacillus were isolated from five heavily contaminated liquid antacids and their constituent chemicals. The 66 different reaction profiles obtained were expressed in probability figures and stored in a computer. A total of 13 Bacillus species were identified, with B. coagulans, B. licheniformis, B. subtilis, and B. polymyxa present at particularly high frequencies. The potential advantage of using a computer in the identification of aerobic sporeformers is demonstrated.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.     

Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR

AIMS: A real-time PCR-based method was developed to evaluate the Bifidobacterium rRNA operon copy number. As a result of their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of heterologous genes. METHODS AND RESULTS: The rrn operon multiplicity per chromosome was determined by real-time PCR quantification of the 16S rRNA amplicons obtained from genomic DNA. The values obtained in several bifidobacterial strains of human origin ranged from 1 to 5. The reliability of the method developed was confirmed by Southern hybridization technique. CONCLUSIONS: In the Bifidobacterium genus the rrn operon copies showed variability at species and strain level. The identification of Bifidobacterium strains with high rRNA multiplicity allowed the selection of potential hosts for chromosomal integration. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology here proposed represents a rapid, reliable and sensitive new tool for the quantification of rrn operon copy number in bacteria.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L. (Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.     

Computer-assisted method for identification of Bacillus species isolated from liquid antacids.

One hundred nineteen species of Bacillus were isolated from five heavily contaminated liquid antacids and their constituent chemicals. The 66 different reaction profiles obtained were expressed in probability figures and stored in a computer. A total of 13 Bacillus species were identified, with B. coagulans, B. licheniformis, B. subtilis, and B. polymyxa present at particularly high frequencies. The potential advantage of using a computer in the identification of aerobic sporeformers is demonstrated.