A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae.

We report the discovery of a group of highly conserved DNA sequences located, in those cases studied, within intergenic regions of the chromosome of the Gram positive Streptococcus pneumoniae. The S. pneumoniae genome contains about 25 of these elements called BOX. From 5' to 3', BOX elements are composed of three subunits (boxA, boxB, and boxC) which are 59, 45 and 50 nucleotides long, respectively. BOX elements containing one, two and four copies of boxB have been observed; boxB alone was also detected in one instance. These elements are unrelated to the two most thoroughly documented families of repetitive DNA sequences present in the genomes of enterobacteria. BOX sequences have the potential to form stable stem-loop structures and one of these, at least, is transcribed. Most of these elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of S. pneumoniae. This location raises the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence-specific and virulence-related genes.     

BOX-PCR技术在微生物多样性研究中的应用

近年来在微生物多样性研究中,利用微生物基因组中广泛分布的短重复序列设计引物,选择性地扩增重复序列之间的不同基因区域,以得到大小不等的DNA扩增片段的方法日渐增多.以BOX插入因子(细菌基因组重复序列)为基础的PCR技术,具有操作简单快捷,可重复性强,容易获得较为丰富的扩增条带等特点,最初主要应用于细菌的多样性研究.目前研究发现用BoxA1R引物对微生物中的真菌、放线菌进行选择性的扩增,也能够达到很好的遗传及多样性分析的目的.本文综述了BOX-PCR指纹图谱分析技术的特点和一般步骤;结合作者对植物内生细菌的BOX-PCR指纹图谱分析体系的优化,对BOX-PCR技术的改进进行了总结:并对该技术在微生物菌株多样性研究领域的应用现状和前景进行了阐述.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

The physiological and genetic diversity of bovine Streptococcus bovis strains(1)

Laboratory Streptococcus bovis strains and isolates obtained from a steer fed increasing amounts of grain had similar growth characteristics, but they differed in their sensitivity to 2-deoxyglucose (2DG), a non-metabolizable glucose analog. The addition of 2DG decreased both growth rate (0.92+/-0.34 h(-1)) and growth yield (ranging from 25 to 63%), but these differences could not be correlated with diet. However, isolates from a steer fed a 90% grain diet were more prone to 2DG-dependent lysis than those from a hay diet (P<0.001). All S. bovis laboratory strains and isolates had an identical restriction fragment length polymorphism pattern, when their 16S rDNA was digested with HaeIII and HhaI. However, when genomic BOX elements were amplified, 5-12 bands were observed, and the S. bovis isolates and laboratory strains could be grouped into 13 different BOX types. Strains 26 and 581AXY2 had the same BOX type, but the remaining laboratory strains did not form closely related clusters. Strains JB1 and K27FF4 were most closely related to each other. Most of the fresh isolates (24 out of 30) could be grouped into a single cluster (>90% Dice similarity). This cluster contained isolates from all three diets, but it did not have any of the laboratory strains. The majority (90%) of the isolates obtained from the hay-fed steer exhibited the same BOX type. Because more BOX types were observed if grain was added to the diet, it appears that ruminal S. bovis diversity may be a diet-dependent phenomenon.     

载有磷酸甘油激酶基因启动子的新表达载体的构建以及在发酵性丝孢酵母中启动外源基因的表达研究

利用简并PCR技术从一株丝孢酵母(Trichosporon sp.)中克隆到磷酸甘油激酶基因的部分序列,然后利用染色体步移的方法克隆到了已知片段的上游序列约950bp。通过启动子序列分析软件分析,发现序列中含有启动子所需的必须元件如TATA BOX和CAAT BOX等,因此确定克隆到的基因片段含有启动子序列。将潮霉素基因置于该启动子下构建了丝孢酵母整合型表达载体pTFPH,并转化发酵性丝孢酵母(Trichosporon fermentans),转化后的酵母能够在含有潮霉素的抗性选择性平板长出,而未进行转化的对照菌株则不能生长。以上试验证明:丝孢酵母的磷酸甘油激酶基因启动子具有启动异源基因在发酵性丝孢酵母中表达的功能,这个结果为油脂酵母工程菌的构建和开发新的酵母表达宿主奠定了基础。     

金属离子对漆斑菌产胆红素氧化酶的影响

比较了漆斑菌在8种液体培养基中胆红素氧化酶(BOX)产量,发现马铃薯液体培养基(PDB)是最适宜漆斑菌产BOX的培养基。研究了8种常见金属离子对漆斑菌产酶的影响,结果表明钠离子、铜离子可以明显促进BOX产量提高,铜离子效果最强,随着铜离子浓度增加,BOX酶产量可进一步提高,但高浓度的铜离子(1mmol.L-1)会抑制酶产量增加。     

Genetic localization of diuron- and mucidin-resistant mutants relative to a group of loci of the mitochondrial DNA controlling coenzyme QH2-cytochrome c reductase in Saccharomyces cerevisiae.

Summary Diuron-resistance, DIU (Colson et al., 1977), antimycin-resistance, ANA (Michaelis, 1976; Burger et al., 1976), funiculosin-resistance, FUN (Pratje and Michaelis, 1977; Burger et al., 1977) and mucidin-resistance, MUC (Subik et al., 1977) are each coded by a pair of genetic loci on the mit DNA of S. cerevisiae. In the present paper, these respiratory-competent, drug-resistant loci are localized relative to respiratory-deficient BOX mutants deficient in coenzyme QH₂-cytochrome c reductase (Kotylak and Slonimski, 1976, 1977) using deletion and recombination mapping. Three drug-resistant loci possessing distinct mutated allelic forms are distinguished. DIU1 is allelic or closely linked to ANA2, FUN1 and BOX1; DIU2 is allelic or closely linked to ANA1, MUC1 and BOX4/5; MUC2 is allelic to BOX6. The high recombinant frequencies observed between the three loci (13% on the average for 33 various combinations analyzed) suggest the existence of either three genes coding for three distinct polypeptides or of a single gene coding for a single polypeptide but subdivided into three easily separable segments. The resistance of the respiratory-chain observed in vitro in the drug-resistant mutants and the allelism relationships between respiratory-competent, drug-resistant loci and coQH₂-cyt c reductase deficient, BOX, loci strongly suggest that each of the three drug-resistant loci codes for a structural gene-product which is essential for the normal coQH₂-cyt c reductase activity and is obviously a good candidate for a gene product of the drug-resistant loci mapped in this paper. Polypeptide length modifications of cytochrome b were observed in mutants deficient in the coQH₂-cyt c red and localized at the BOX1, BOX4 and BOX6 genetic loci (Claisse et al., 1977, 1978) which are precisely the loci allelic to drug resistant mutants as shown in the present work. Taken together these two sets of data provide a strong evidence in favor of the idea that there exist three non contiguous segments of the mitochondrial DNA sequence which code for a single polypeptide sequence of cytochrome b. In each segment mutations which modify the polypeptide sequence can occur leading to the loss (BOX mutants) or to a modification (drug resistant mutants) of the enzyme activity.Chercheur qualifié du Fonds National de la Recherche Scientifique     

Genotypic and phenotypic diversity of PGPR fluorescent pseudomonads isolated from the rhizosphere of sugarcane (Saccharum officinarum L.)

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.     

A new tactic for the treatment of jaundice: an injectable polymer-conjugated Bilirubin oxidase

Bilirubin oxidase (BOX) derived from Myrothecium verrucaria was modified with polyethylene glycol (PEG). When the conjugated PEG-BOX was given intravenously to rats, its plasma half-life was 20 times longer than that of native BOX. In our preliminary investigations with experimentally jaundiced rats, the plasma bilirubin level dropped to normal after only one injection, and the low bilirubin level could be maintained for 12-48 hr; native BOX had a transitory suppressive effect that lasted only a few hours. The antigenicity of PEG-BOX was greatly reduced as expected. PEG-BOX appears to have potential value for the treatment of hyperbilirubinemia observed in such diseases as fulminant hepatitis and neonatal bilirubin encephalopathy.     

Use of repetitive DNA sequences and the PCR To differentiate Escherichia coli isolates from human and animal sources

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.     

Genetic diversity of Ralstonia solanacearum strains from China assessed by PCR-based fingerprints to unravel host plant- and site-dependent distribution patterns

Bacterial wilt caused by Ralstonia solanacearum is a serious threat to crop production in China. A collection of 319 R. solanacearum strains isolated from 14 different diseased host plants collected in 15 Chinese provinces was investigated by BOX fingerprints in order to test the influence of the site and the host plant on their genetic diversity. Phylotype, fliC-RFLP patterns and biovar were determined for all strains and the sequevar for 39 representative strains. The majority of strains belonged to the Asian phylotype I, shared identical fliC-RFLP patterns and were assigned to four biovars (bv3:123; bv4:162; bv5:3; and bv6:11). Twenty strains were phylotype II, assigned to biovar 2, and had distinct fliC-RFLP patterns. BOX-PCR fingerprints generated from the genomic DNA of each strain revealed a high diversity of the phylotype I strains, where 28 types of BOX fingerprints could be distinguished. While many BOX clusters comprised isolates from different provinces and several host plants, some groups contained isolates that were plant or site specific. All phylotype II isolates originating from 10 provinces belonged to sequevar 1 and displayed identical BOX patterns as the potato brown rot strains from various regions of the world.     

Use of Repetitive DNA Sequences and the PCR To Differentiate Escherichia coli Isolates from Human and Animal Sources

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.     

Characterisation of micromonosporae from aquatic environments using molecular taxonomic methods

Large numbers of strains assigned to the genus Micromonospora on the basis of typical colonial and pigmentation features were isolated from diverse aquatic sediments using a standard selective isolation procedure. Two hundred and six isolates and eight representatives of the genus Micromonospora were assigned to 24 multimembered groups based on a numerical analysis of banding patterns generated using BOX and ERIC primers. Representatives of multimembered groups encompassing isolated micromonosporae were the subject of 16S rRNA gene sequencing analyses. Good congruence was found between the molecular fingerprinting and 16S rRNA sequence data indicating that the groups based upon the former are taxonomically meaningful. Nearly all of the isolates that were chosen for the 16S rRNA gene sequencing analyses showed that the ecosystems studied are a rich source of novel micromonosporae. These findings have implications for high throughput screening for novel micromonosporae as BOX and ERIC fingerprinting, which is rapid and reproducible, can be applied as a robust dereplication procedure to indicate which environmental isolates have been cultured previously.     

魔芋软腐病菌分子鉴定与遗传多样性

通过对分离的魔芋软腐病菌株和其它参试菌株的致病性测定、选择性培养基培养性状观察和16S-23S rDNA转录间隔区PCR(ITS-PCR)分析,将测试的33株软腐病菌株主要分为3个组群。第1组群为胡萝卜软腐欧文氏杆菌胡萝卜软腐亚种(Erwinia carotovorasubsp.carotovora,E.c.c.);第2组群为菊欧文氏杆菌(Erwinia chrysanthemi,E.ch.);还有一组未能确定的菌株。利用细菌基因组重复序列通用引物BOX和J3进行Rep-PCR特异性扩增,引起软腐病的菌株E.c.c.和E.ch.(ITS-PCR鉴定)种内的Rep-PCR指纹存在明显的遗传分化,经聚类分析,在0.1水平上把E.c.c.13株区分为5个类群。     

Differentiation of lineages within “Colletotrichum gloeosporioides s.l.” associated with cassava anthracnose disease by BOX‐ and ERIC‐PCRs

Several molecular techniques have been used to differentiate species or genetic lineages of microorganisms prior to sequencing. Among them, BOX‐ and ERIC‐PCRs may provide specific banding patterns for different species, allowing its differentiation. Therefore, the objective of this study was to evaluate these techniques as a tool for differentiation of phylogenetic lineages belonging to the Colletotrichum gloeosporioides species complex associated with cassava anthracnose disease. Sets of BOX‐ and ERIC‐PCR primers were used to assess the differentiation of lineages belonging to the complex with 81 C. gloeosporioides sensu lato (s.l.) isolates from different cassava producing regions. Some were identified by sequencing, such as Colletotrichum fructicola, Colletotrichum tropicale, C. gloeosporioides s.s, Colletotrichum theobromicola, Colletotrichum siamense, Colletotrichum brevisporum and Colletotrichum sichuanensis. The primers were able to amplify DNA fragments from all isolates. The ERIC‐PCR presented a wider range of banding patterns in comparison to BOX‐PCR, providing better differentiation of the individuals, as well as a higher correlation with the phylogenetic data was obtained by ERIC‐PCR and the combined data set for “BOX‐/ERIC‐PCRs,” inferred by Mantel test. However, the use of concatenated data (BOX‐/ERIC‐PCRs) reduced the discriminatory capacity presented by ERIC‐PCR alone, probably due to the lowest resolution of BOX‐PCR. Therefore, ERIC‐PCR technique enabled efficient differentiation of isolates belonging to the C. gloeosporioides complex and can be used to analyse multiple isolates in a collection and also being an important tool as a guide in the decision‐making process prior to sequencing. Based on this methodology, it was possible to identify two new species associated with cassava anthracnose disease, C. brevisporum and C. sichuanensis, being the first report of these two species associated with cassava anthracnose disease in Brazil.     

Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.