An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups

Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.     

A System of Oligonucleotide Primers for the Amplification of nifH Genes of Different Taxonomic Groups of Prokaryotes

Based on the analysis of the nifH gene nucleotide sequences from GenBank, a system of primers was developed that makes it possible to obtain 370- and 470-bp PCR fragments of the nifH gene of nitrogen-fixing bacteria and archaea. The effectiveness of the proposed system for revealing the presence of nifH genes was demonstrated by PCR on the DNA isolated from nitrogen-fixing prokaryotes for which the primary structure of these genes is known and which belong to different taxonomic groups. nifH sequences of nitrogen-fixing prokaryotes of the genera Xanthobacter, Beijerinckia, and Methanosarcina, for which the capacity for nitrogen fixation was demonstrated earlier, but no data existed on the nucleotide composition of these genes, were determined and deposited in GenBank.     

A Study of Nucleotide Sequences of nifH Genes of Some Methanotrophic Bacteria

Using a previously developed primer system, nifH gene fragments 450 nucleotides long were amplified, cloned, and sequenced for representatives of nitrogen-fixing methanotrophic bacteria of the genera Methylococcus, Methylocystis, and Methylosinus. Fragments of nifH genes were also detected and sequenced in representatives of the genera Methylomonas and Methylobacter, which were not considered diazotrophs until recently. Phylogenetic analysis revealed the remoteness of nifH gene sequences of methanotroph types I and II. At the same time, a close relationship was found between nifH of type I methanotrophs and representatives of -proteobacteria and between nifH genes of type II methanotrophs and representatives of -proteobacteria. The results obtained in this study are in good accordance with the data of phylogenetic analysis based on 16S rRNA sequence comparison with the only exception being Methylococcus capsulatus strains, whose nifH genes proved to be closely related to nifH genes of Methylocystis and Methylosinus representatives. Our findings extend the database of primary sequences of nifH genes and allow the contribution of methanotrophs to the process of nitrogen fixation to be estimated.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.Abbreviations RFLP Restriction fragment length polymorphism - MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight spectrometry - HPLC High performance liquid chromatography     

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.     

Detection of a novel spotted fever group rickettsia in <Emphasis Type="Italic">Amblyomma parvum</Emphasis> ticks (Acari: Ixodidae) from Argentina

The present study evaluated the rickettsial infection in Amblyomma parvum ticks collected in Northwestern Córdoba Province, Argentina. Each tick was subjected to DNA extraction and tested by polymerase chain reaction (PCR) targeting fragments of the rickettsial genes gltA and ompB. Nine (69.2%) out of 13 adult ticks yielded expected PCR products for the two rickettsial genes. Products from the ompB PCR were sequenced, generating DNA sequences 100% identical for the nine PCR-positive ticks. Three of these ticks were tested in another battery of PCR targeting fragments of the rickettsial genes gltA, htrA, and ompA. Products from the gltA, htrA, and ompA PCRs were sequenced generating DNA sequences 100% identical for the three PCR-positive ticks. The rickettsia detected in the A. parvum ticks was designated as Rickettsia sp. strain Argentina. Phylogenetic analyses performed with partial sequences of the rickettsial genes gltA, htrA, ompB, and ompA showed that Rickettsia sp. strain Argentina belonged to the spotted fever group, being distinct from all known Rickettsia species and genotypes available in GenBank, representing possibly a new Rickettsia species. This was the first evidence of rickettsial infection in the tick A. parvum, and the third report of rickettsial infection among the Argentinean tick fauna. The role of Rickettsia sp. strain Argentina as a human pathogen is unknown. Further studies are needed to obtain tissue-cultured isolates of Rickettsia sp. strain Argentina, in order to better characterize it and to determine its taxonomic status as a new species.     

Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the <Emphasis Type="Italic">amoCAB</Emphasis> operon

The genes and intergenic regions of the amoCAB operon were analyzed to establish their potential as molecular markers for analyzing ammonia-oxidizing betaproteobacterial (beta-AOB) communities. Initially, sequence similarity for related taxa, evolutionary rates from linear regressions, and the presence of conserved and variable regions were analyzed for all available sequences of the complete amoCAB operon. The gene amoB showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used amoA to resolve closely related AOB species. To test the suitability of using the amoCAB genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole amoCAB operon and each individual gene were tested. The specificity of the products generated was analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The fragments obtained showed different grades of sequence identity to amoCAB sequences in the GenBank database. The nested PCR approach provides a possibility to increase the sensitivity of detection of amo genes in samples with low abundance of AOB. It also allows the amplification of the almost complete amoA gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three amo genes and the intergenic spacer regions that are under different selection pressure might allow a more detailed analysis of the evolutionary processes, which are responsible for the differentiation of AOB communities in different habitats. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Pilar Junier and Ok-Sun Kim contributed equally to this work.     

Molecular Discrimination of Perna (Mollusca: Bivalvia) Species Using the Polymerase Chain Reaction and Species-Specific Mitochondrial Primers

This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species-specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working.     

Molecular cloning and characterization of a profilin gene <Emphasis Type="Italic">BnPFN</Emphasis> from <Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">nigra</Emphasis> that expressing in a pollen-specific manner

Brassica nigra is a newly found invasive species in Zhejiang Province, China. It distributes alongside the roads, in vegetable fields and on riversides. When it blooms, some natives there will suffer from allergic rhinitis. We designed gene-specific primer pairs according to reported profilin genes and successfully isolated their homolog from flower bud cDNA of B. nigra. The gene, designated BnPFN, was submitted to GenBank under accession number EU004073. BnPFN was 405 bp in length encoding 134 amino acids. Expression analysis of BnPFN gene was carried out by means of RT-PCR. The results showed that BnPFN express only in anthers and pollens, and there was no detection in roots, leaves, stems, sepals, petals and pistils. We suggest that BnPFN is a pollen-specific gene and may be responsible for pollen anaphylactic reactions in those invading areas when B. nigra blooms.     

珙桐内生细菌的分离鉴定及系统发育分析

用微生物学传统分离培养方法从珙桐茎、叶及叶柄中分离到内生细菌56株,选取17株内生菌,提取其基因组DNA,并以此为模板,PCR扩增其16S rDNA,扩增出约1 500 bp大小的DNA条带,对15株内生菌16S rDNA测序,用BLAST软件对测序结果进行相似性比对,发现11株为Bacillus属,相似性为95%-98%;2株为Lysinibacillus属,相似性为97%和99%;1株为Bordetella属,相似性为95%;1株为非培养细菌的同源菌,相似性96%。芽孢杆菌为珙桐内生细菌优势菌属。通过构建系统发育树发现这15株内生菌明显聚为2大支。     

Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers and gene mapping based on SNPs

The Dreb genes are involved in abiotic stress tolerances, such as drought, salinity, low temperature and ABA. The purpose of the present research was to establish protocols for the development of genome-specific and allele specific markers in common wheat (Triticum aestivum L.) using the Dreb1 genes as an example. Based on the available sequences of Dreb1 genes in common wheat and related species, five primer pairs were designed using Primer Premier 5.0. Two primers, P25F/PR and P21F/P21R, amplified 596- and 1113-bp fragments, respectively, from the A genome, P18F/P18R amplified a 717-bp fragment from the B genome, and primers P22F/PR and P20F/P20R amplified 596- and 1193-bp fragments, respectively, from the D genome. Using these genome-specific primers and the Chinese Spring using nulli-tetrasomic lines, the Dreb1 genes were located on chromosomes 3A, 3B and 3D. Two SNPs (S646 and S770) in Dreb-B1 distinguished the Opata 85 and W7984 parents of the ITMI mapping population, but there was no polymorphism between the orthologous Dreb-A1 and Dreb-D1 sequences. By assaying the genotypes of 115 RILs with the allele-specific primer P40 based on SNP S770, Dreb-B1 was mapped between markers Xmwg818 and Xfbb117 on chromosome 3BL. This genetic mapping of Dreb-B1 on chromosome 3B may be helpful in wheat breeding programs aimed at improving drought tolerance.     

Development of “universal” gene-specific markers from Malus spp. cDNA sequences, their mapping and use in synteny studies within Rosaceae

The Rosaceae contains many economically valuable crop genera, including Malus (apple), Fragaria (strawberry), and Prunus (stone fruit). There has been increasing interest in the development of linkage maps for these species, with a view to marker-assisted selection to assist breeding programs and, recently, in the development of transferable markers to permit syntenic comparisons of maps of different rosaceous genera. In this investigation, a set of Malus cDNA sequences were downloaded from the European Molecular Biology Laboratory database. The sequences were aligned with homologous full-length Arabidopsis genomic DNA sequences to identify putative intron–exon junctions and conserved flanking exon sequences. Primer pairs were designed from the conserved exon sequences flanking predicted intron–exon junctions in the Malus cDNA sequences. These were used to amplify products by polymerase chain reaction from the parents of the Malus mapping progeny “Fiesta” × “Totem.” Eleven loci, representing ten genes (39%), were polymorphic in the “Fiesta” × “Totem” population and mapped to seven Malus linkage groups. Transferability to other rosaceous genera was high, with primer pairs representing 85% of genes, amplifying products from Fragaria and primer pairs representing 85% of genes, amplifying products from Prunus genomic DNA. These primers were screened in the Fragaria and Prunus mapping bin sets and 38% of the genes were successfully located on both maps. Analysis of the markers mapped in more than one rosaceous genus revealed patterns of synteny between genera, while a comparison with the physical positions of homologous genes on the Arabidopsis genome revealed high sequence conservation but only fragmentary patterns of macrosynteny.     

Identification of mycorrhiza-regulated genes with arbuscule development-related expression profile

Suppressive subtractive hybridisation was applied to the analysis of late stage arbuscular mycorrhizal development in pea. 96 cDNA clones were amplified and 81, which carried fragments more than 200 nt in size, were sequence analysed. Among 67 unique fragments, 10 showed no homology and 10 were similar to sequences with unknown function. RNA accumulation of the corresponding 67 genes was analysed by hybridisation of macro-arrays. The cDNAs used as probes were derived from roots of wild type and late mutant pea genotypes, inoculated or not with the AM fungus Glomus mosseae. After calibration, a more than 2.5-fold mycorrhiza-induced RNA accumulation was detected in two independent experiments in the wild type for 25 genes, 22 of which seemed to be induced specifically during late stage AM development. Differential expression for 7 genes was confirmed by RT-PCR using RNA from mycorrhiza and from controls of a different pea cultivar. In order to confirm arbuscule-related expression, the Medicago truncatula EST data base was screened for homologous sequences with putative mycorrhiza-related expression and among a number of sequences with significant similarities, a family of trypsin inhibitor genes could be identified. Mycorrhiza-induced RNA accumulation was verified for five members by real-time PCR and arbuscule-related activation of the promoter could be shown in transgenic roots for one of the genes, MtTi1.     

Fusarium oxysporim M1菌株的rDNA ITS鉴定及其纤维素酶活性及纯化研究

以1株分离于北大仓白酒大曲的产纤维素酶真菌M1为材料,对其进行了形态及分子生物学鉴定;纯化并研究了其纤维素酶的酶学性质。以真菌ITS1/ITS4通用引物,扩增真菌M1的rDNA ITS序列,再与GenBank中其他菌株rDNA ITS序列进行比对,使用Mega5.0软件,采用最大似然法进行聚类分析。结果显示,该真菌同已经报道的Fusarium oxysporim strain Bt3聚为一类,一致率达99%,与形态学方法鉴定一致,命名为Fusarium oxysporum M1。该菌具有很高纤维素酶活力,FPA和CMCA分别高达16.84 IU/mL和35.31 IU/mL。经过发酵条件优化酶活性进一步提高。经硫酸铵分级分离、疏水和离子交换层析,纯化了该菌纤维素酶,纯化倍数高达17.97倍,得率为3.676%,SDS-PAGE分析表明,该纤维素酶分子量达60 k Da。本研究为进一步研究该酶高效催化机理及实际应用提供参考。     

Phylogeny of anoxygenic filamentous phototrophic bacteria of the family Oscillochloridaceae as inferred from comparative analyses of the rrs, cbbL, and nifH genes

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae (Oscillochloris trichoides DG6T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA (rrs), ribulose- 1,5-bisphosphate carboxylase/oxygenase (cbbL), and nitrogenase (nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the "red-like" form I RuBPC were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their identification as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes.     

Classification of <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. using rDNA-ITS sequence analysis supports the genetic basis of the classical anastomosis grouping

Currently, rDNA-ITS sequence analysis seems to be the most appropriate method for comprehensive classification of Rhizoctonia spp. Our previous review article was concerned with detailed analysis of multinucleate Rhizoctonia (MNR), and the current review complements the previous one with detailed analysis of binucleate Rhizoctonia (BNR) (teleomorphs: Ceratobasidium spp. and Tulasnella spp.) and uninucleate Rhizoctonia (UNR) (teleomorph: C. bicorne). Data of all the appropriate BNR and UNR accumulated in GenBank were analyzed together in neighbor-joining (NJ) trees supplemented with percent sequence similarity within and among the anastomosis groups (AGs) and subgroups. Generally, the clusters of the isolate sequences supported the genetic basis for the AG based on hyphal fusion anastomosis. Comprehensive interrelationships among all the currently available MNR, BNR, and UNR groups and subgroups in GenBank were subsequently analyzed in NJ and maximum-parsimony (MP) trees, showing the genetic relatedness among the different groups and indicating possible bridging groups between MNR, BNR, and UNR. The review also indicates serious inaccuracies in designation of sequences of some isolates deposited in GenBank. Several additional teleomorph genera with Rhizoctonia spp. anamorphs have also been reported in the literature. However, as they have not been intensively studied, there were no available data on their rDNA-ITS sequences that could be included in this review.     

Characterization of a new organization of the plantaricin locus in the inducible bacteriocin-producing<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> J23 of grape must origin

Lactobacillus plantarum J23 was previously characterized as a bacteriocin-producer-strain when it was cocultured with other lactic acid bacteria. In this work, the genetic organization of the pln locus in the J23 strain was studied and compared with those of previously described L. plantarum C11, WCFS1 and NC8 strains. A new organization of the plantaricin locus was detected in the J23 strain. The sequenced fragment (20,266 bp) comprised plnJLR, plnMNOP, plnEFI, plnGHSTUVWXY, and plNC8IF-plNC8HK-plnD operons, as well as a new region that includes three new orfs (GenBank accession number DQ323671). When the J23 pln gene sequences were compared with those included in the GenBank database, the identity of the putative encoded proteins was in the range 67.1–100%. The regulatory system and the repertoire of putative bacteriocins of the J23 pln locus presented important differences with respect to the ones of C11, WCFS1 and NC8, such as the absence of plnK and the presence of a larger plnJ gene than the previously described for the other L. plantarum strains. The pln locus in L. plantarum strains seems to be a mosaic-like structure with different modules and reorganizations that presents highly conserved regions related to transport and bacteriocin maturation and variable regions related to regulation and bacteriocin production.     

Mhc class I genes of the cichlid fish Oreochromis niloticus

In terms of number of species, perciform (perch-like) fishes are one of the most diversified groups of modern vertebrates. Within this group, the family Cichlidae is best known for its spectacular adaptive radiation in the great lakes of East Africa. The molecular tool kit used in the study of this radiation includes the major histocompatibility complex (Mhc) genes. To refine this tool, information about the organization of the Mhc regions is badly needed. In this study, the first step was taken toward providing such information for the Mhc class one regions of Oreochromis niloticus, a representative species of the tilapiine branch of the Cichlidae, for which good bacterial artificial chromosome library is available. Screening of the library with class I gene probes led to the identification and isolation of 31 class-I-positive clones. Sequencing of one of these clones and partial characterization of the remaining clones for the presence of class I exons resulted in the construction of two contigs representing the class I region of this species as well as identification of seven additional class-I-positive singleton clones. The O. niloticus genome was shown to contain at least 28 class I genes or gene fragments. The shorter of the two contigs was approximately 330 kb long and contained eight class I genes/gene fragments; the longer contig encompassed 1,200 kb of sequence and contained minimally 17 class I genes/gene fragments; three additional class I genes were found to be borne by a clone that might be part of the shorter contig. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. This work had been carried out in part at the Max-Planck-Institut für Biologie, Abteilung Immungenetik, Tübingen, Germany (A.S., R.D., N.T., S.S., and J.K.). The sequences reported in this paper have been deposited in the GenBank database (accession nos. AB270803–AB270897).     

System of oligonucleotide primers for detection and amplification of the emoA gene encoding bacterial ethylenediaminetetraacetate monooxygenase

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoAgene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoAgene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.