Identification and application of plasmids suitable for transfer of foreign DNA to members of the genus Gordonia

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 x 10(5) CFU/ micro g of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499(T), G. rubropertincta DSM43197(T), G. rubropertincta DSM46038, and G. terrae DSM43249(T). Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 x 10(-6) of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed.     

Selective Isolation and Rapid Identification of Members of the Genus Micromonospora

Improved methods for selective isolation of diverse actinomycetes of the genus Micromonospora and a genus-specific nested PCR for rapid identification of putative Micromonospora isolates were developed. The robustness of both the isolation and the identification approach was underpinned by phylogenetic analysis based on 16S rRNA gene sequences.     

Genetic Identification of Members of the Genus Corynebacterium at Genus and Species Levels with 16S rDNA-Targeted Probes

16S rRNA gene-targeted probes were designed for the identification of corynebacteria at the genus and species levels. The genus-specific probe hybridized all clinically important members of the genus Corynebacterium and could distinguish them from other coryneform bacteria and phylogenetically related high G + C% gram-positive bacteria, including Actinomyces, Rhodococcus, Gordona, Nocardia, Streptomyces, Brevibacterium and Mycobacterium. The species-specific probes for C. jeikeium and C. diphtheriae could differentiate these two species from other members of this genus. The probes were used to select corynebacteria among gram-positive clinical isolates which had been tentatively identified as corynebacteria by biochemical tests. We screened 59 strains with the genus-specific probe; 51 strains hybridized to the genus-specific probe, 8 did not. Of the 51 strains that hybridized to the genus-specific probe, 1 hybridized to the C. diphtheriae species probe and 13 hybridized to the C. jeikeium species probe. The 8 strains that did not hybridize to the genus probe were further characterized by analyzing cell wall diaminopimelic acid and partial 16S rRNA sequencing. The results indicated that these strains were distributed in the genera Arthrobacter and Brevibacterium.     

Identification of the Mima-Herellea Group of Organisms and Selected Members of the Genus Neisseria

To determine a rapid and reliable protocol for the differentiation of Mima polymorpha, M. polymorpha var. oxidans, Herellea, Bacterium antitratum, Neisseria gonorrheae, and other related members of the genus Neisseria, reference cultures were examined on a variety of microbiological media. The media were selected because of the typical morphological and biochemical characteristics exhibited by the test organisms and were those media which would be readily available in a microbiology facility. After compiling data obtained from 21,714 observations, an inclusive protocol is presented which has proven to be quite adequate for the identification of these particular gram-negative bacteria.     

一种用质粒DNA转化大肠杆菌感受态细胞的实用操作技巧

目的是建立一种简化、实用的用质粒DNA转化大肠杆菌的操作方法.采用氯化钙法制备大肠杆菌感受态细胞.以质粒pUC18,pCSN44,pAN52-1Not,pETts,pANth和植物双元表达栽体pCAMBIA1301分别转化用于质粒扩增与保存的常用大肠杆菌菌株Top10和DH5α以及用于原核表达的常用大肠杆菌菌株BL21(DE3)和TB1.质粒与感受态细胞的混合液置冰上作用一定时间后,直接涂布含有筛选抗生素的LB平板,于37℃培养12～16h.结果表明,用不同大小的质粒DNA转化不同的大肠杆菌菌株,都可以获得满足实验要求,转化效率可高达103～4阳性克隆/μg.该方法较标准的转化流程更加简便、省时、实用.     

Identification of Two Novel Members of the Tentative Genus Wukipolyomavirus in Wild Rodents

Two novel polyomaviruses (PyVs) were identified in kidney and chest-cavity fluid samples of wild bank voles (Myodes glareolus) and common voles (Microtus arvalis) collected in Germany. All cloned and sequenced genomes had the typical PyV genome organization, including putative open reading frames for early regulatory proteins large T antigen and small T antigen on one strand and for structural late proteins (VP1, VP2 and VP3) on the other strand. Virus-like particles (VLPs) were generated by yeast expression of the VP1 protein of both PyVs. VLP-based ELISA and large T-antigen sequence-targeted polymerase-chain reaction investigations demonstrated signs of infection of these novel PyVs in about 42% of bank voles and 18% of common voles. In most cases only viral DNA, but not VP1-specific antibodies were detected. In additional animals exclusively VP1-specific antibodies, but no viral DNA was detected, indicative for virus clearance. Phylogenetic and clustering analysis including all known PyV genomes placed novel bank vole and common vole PyVs amongst members of the tentative Wukipolymavirus genus. The other known four rodent PyVs, Murine PyV and Hamster PyV, and Murine pneumotropic virus and Mastomys PyV belong to different phylogenetic clades, tentatively named Orthopolyomavirus I and Orthopolyomavirus II, respectively. In conclusion, the finding of novel vole-borne PyVs may suggest an evolutionary origin of ancient wukipolyomaviruses in rodents and may offer the possibility to develop a vole-based animal model for human wukipolyomaviruses.     

Monoclonal Antibodies Specific for Members of the Genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni , M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas. Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni , M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64–200 kD.     

Comparative Analysis of Plasmids in the Genus Listeria

Background

We sequenced four plasmids of the genus Listeria, including two novel plasmids from L. monocytogenes serotype 1/2c and 7 strains as well as one from the species L. grayi. A comparative analysis in conjunction with 10 published Listeria plasmids revealed a common evolutionary background.

Principal Findings

All analysed plasmids share a common replicon-type related to theta-replicating plasmid pAMbeta1. Nonetheless plasmids could be broadly divided into two distinct groups based on replicon diversity and the genetic content of the respective plasmid groups. Listeria plasmids are characterized by the presence of a large number of diverse mobile genetic elements and a commonly occurring translesion DNA polymerase both of which have probably contributed to the evolution of these plasmids. We detected small non-coding RNAs on some plasmids that were homologous to those present on the chromosome of L. monocytogenes EGD-e. Multiple genes involved in heavy metal resistance (cadmium, copper, arsenite) as well as multidrug efflux (MDR, SMR, MATE) were detected on all listerial plasmids. These factors promote bacterial growth and survival in the environment and may have been acquired as a result of selective pressure due to the use of disinfectants in food processing environments. MDR efflux pumps have also recently been shown to promote transport of cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger a cytosolic host immune response following infection.

Conclusions

The comparative analysis of 14 plasmids of genus Listeria implied the existence of a common ancestor. Ubiquitously-occurring MDR genes on plasmids and their role in listerial infection now deserve further attention.     

Plasmids of the Chlorophenoxyacetic-Acid Degradation of Bacteria of the Genus Raoultella

Applied Biochemistry and Microbiology - Members of the Raoultella planticola species isolated from soils contaminated with chemical waste were able to use...     

抗砷载体的构建及在喜温硫杆菌中的接合转移

利用DNA体外重组技术,将质粒载体pUM3上的抗砷基因片段亚克隆到含有强启动子(tae启动子)并具有广泛寄主范围特性的IncQ族质粒pMMB24上,成功构建了含有强启动子的抗砷质粒pSDRA3,以及删除调节基因片段的组成型表达的抗砷质粒pSDRA4。通过接合转移的方式将其导入专性自养极端嗜酸性的喜温硫杆菌中,首次成功地建立了喜温硫杆菌的遗传转移系统,构建了冶金工程菌T.caldus(pSDRA3)和T．caldus(pSDRA4)。与野生菌相比,重组菌抗砷性能明显提高。     

Transfer of IncP Plasmids to Extremely Acidophilic Thiobacillus thiooxidans

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10^-5 to 10^-7 per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 × 10^-3 per recipient.     

Real-Time PCR for the Detection and Quantification of Geodermatophilaceae from Stone Samples and Identification of New Members of the Genus Blastococcus

Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus.     

高粱属A-PAGE 图谱的建立及在种子鉴定中的应用

以高粱、苏丹草等5种高粱属植物的种子为材料,利用酸性聚丙烯酰胺凝胶电泳(A-PAGE)检测其醇溶蛋白.同时研究了丙酮处理、样品上样量和凝胶浓度对电泳的影响.结果表明:样品经丙酮处理后,在上样量为32μg、凝胶质量分数为15%时电泳可以得到清晰的A-PAGE图谱,据此可以鉴定5种高粱属植物.     

PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora.     

Evaluation of Comparative DNA Amplification Fingerprinting for Rapid Species Identification within the Genus Clusia

Understanding the taxonomiy of the tropical genus Clusia (Fam. Clusiaceae, Ord. Theales) has been hampered by the difficulties inherent in studying tropical dioecious, succulent, arborescent, epi- or hemiepiphytic taxa. Species identification by morphological traits often requires the terminal inflorescences and/or the succulent capsular fruits. To allow species differentiation based exclusively on vegetative tissue, a frequent necessity during ecological field studies, a procedure has been developed for rapid isolation of genomic DNA from Clusia leaf tissue followed by DNA amplification fingerprinting with a set of single arbitrary oligomer primers (23–27 mers). Fingerprints obtained with independent DNA preparations from one individual as well as DNA preparations from several individuals of the same species were identical for the major amplification products, although minor bands were somewhat variable. Polymorphic fingerprints have been obtained with 3 different primers for 3 Clusia species (C. minor L., C. alata Pl. & Tr., C. multiflora H. B. K.), and the related Oedematopus obovatus Spruce ex. PL (Clusiaceae). The interspecific Randomly Amplified Polymorphic Markers (RAPDs) thus obtained allow a rapid identification of vegetative tissue samples collected in the field, and will assist in a revision of the controversial taxonomy of the genus Clusia.     

Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis

Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis.     

Fungal Metabolite from Members of the Genus Rhizopus

A fluorescent metabolite present in seven members of the genus Rhizopus was isolated. This compound appeared green before spray treatment and purple after spray treatment with p-anisaldehyde in visible light. Subsequent purification and structural elucidation of the isolated compound yielded 1-[2,6,10,14-tetramethyl-17-carbomethyl heptadecyl]-1-[2,6,10,14-tetramethyl-17-methanoyl heptadecyl]-benzene.     

Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using ³²P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T_d) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T_ds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.