Construction and characterization of a cosmid of Streptomyces lividans

Summary We constructed a plasmid pR4C1 in which a DNA fragment containing the cohesive ends of an actinophage, R4 was inserted into the ClaI site of plasmid pIJ365. The plasmid pR4C1 was packaged efficiently into R4 phage particles in vivo and therefore transferred by transduction. Southern hybridization analysis of DNA from pR4C1-transducing R4 phage particles indicated that the plasmid DNA was encapsidated as head-to-tail concatemers with the cohesive ends in the termini. We designated the pR4C1 plasmid a cosmid, following the termination of Collins and Hohn (1978).     

Transduction of a Plasmid with an Inserted R4 Phage DNA Fragment in Streptomyces lividans

A Streptomyces plasmid, pR4C2, with an inserted DNA fragment of R4 phage, was encapsidated into R4 phage particles in vivo and transduced to Streptomyces lividans at 3 ×10^?6CFIJ/PFU. Formation of transducing phage was dependent on the inserted R4 DNA, and some of the transducing phages had larger DNA than R4 phage. A possible transduction mechanism through plasmid-phage cointegrate formation in vivo is discussed.     

Effect of Temperature on the Activities of Cytochrome c Oxidase and Respiration in Cassava Root Mitochondria

Cosmid pR4Cl is a derivative of multicopy plasmid pIJ365 which has an insertion of the cos (cohesive end site) region of actinophage R4 [T. Morino, H. Takahashi and H. Saito, Mol. Gen. Genet., 198, 228 (1985)]. When the donor R4 phage was propagated in S. lividans carrying the plasmid, the phage lysate contained transducing particles which encapsulated head-to-tail concatemers of the plasmid DNA. These particles could mediate transfer of the plasmid at a high frequency. We examined conditions that gave a maximum transduction frequency of cosmid pR4Cl. Conditions which depress R4 phage propagation, such as incubation of recipient S. parvulus at a high temperature, improved the frequency. Obviously such conditions minimized the lethal effect of viable phage propogation. The highest transduction frequency obtained so far was around 3 × 10^-3 transductants per infected phage when S. lividans was used as the recipient. This was about 30 per cent of the cosmid transducing particles estimated from the cosmid DNA content in the transducing lysate. The significance of cosmid transduction for gene manipulation in Streptomyces strains is also discussed.     

Interspecific transfer and expression of melanin gene(s) on cosmids in Streptomyces strains

Summary Cosmid pR4C1 has been constructed from a multicopy plasmid pIJ365 and the cohesive ends site of an actinophage R4 (Morino et al. 1985). The cosmid can be transferred to various Streptomyces strains by R4 phage-mediated transduction. Melanin-synthesizing gene(s) originated from Streptomyces antibioticus was inserted into the cosmid and succesfully transferred by tranduction to 7 different strains out of 10 strains examined. The features of melanin-gene(s) expresion were examined in those cells. Tyrosinase activities from the melanin-gene(s) were detected in culture media and/or cell extracts although the extents of mel gene expression and tyrosinase excretion vary strain to strain. The cosmid transfer system described in this paper will be promising to survey suitable hosts for the maximum expression of cloned genes among Streptomyces strains.     

An active photoreceptor intermediate revealed by in situ photoirradiated solid-state NMR spectroscopy

A novel, to our knowledge, in situ photoirradiation system for solid-state NMR measurements is improved and demonstrated to successfully identify the M-photointermediate of pharaonis phoborhodopsin (ppR or sensory rhodopsin II), that of the complex with transducer (ppR/pHtrII), and T204A mutant embedded in a model membrane. The ¹³C NMR signals from [20-¹³C]retinal-ppR and ppR/pHtrII revealed that multiple M-intermediates with 13-cis, 15-anti retinal configuration coexisted under the continuously photoirradiated condition. NMR signals observed from the photoactivated retinal provide insights into the process of photocycle in the ppR/pHtrII complex.     

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

Summary Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.     

铬黄圆痕叶蝉属的订正及圆痕叶蝉族巴西二新属二新种描记（半翅目：叶蝉科）

基于比较形态学对曾归入圆痕叶蝉亚科铬黄圆痕叶蝉属Chromagallia的8个有效种（C. saucia (St?l), C. flavofasciata (St?l), C. longistilata (Coelho & Dutra), C. carvalhoi Gon?alves et al., C. lamasi Gon?alves et al., C. lanceolata Gon?alves et al., C. paraguayensis Gon?alves et al.和C. zanolae Gon?alves et al.）进行了订正,明确了该属的范围仅限于具有黄斑的3个种（C. flavofasciata (St?l), C. longistilata (Coelho & Dutra) 和C. rodriguesoi sp. nov.）,对该属进行了重新描述,并修订了鉴别特征。此外,对模式种C. flavofasciata 进行了重新描记,首次提供了C. longistilata的雌性生殖器图,并作了描记。把曾归入铬黄圆痕叶蝉属Chromagallia具有红斑的6个种移出并新建了2个新属：Rubragallia 和Neorubragallia,其中Rubragallia 包括R. saucia (St?l) n. comb.和R. paraguayensis (Gon?alves et al.) n. comb., Neorubragallia包括N. lamasi (Gon?alves et al.) n. comb., N. lanceolata (Gon?alves et al.) n. comb., N. zanolae (Gon?alves et al.) n. comb., N. carvalhoi (Gon?alves et al.) n. comb. 和N. mervini sp. nov.。文中提供了3个属的分种检索表,并对不同种的分类地位及3个属的划分进行了讨论。     

Weakened coupling of conserved arginine to the proteorhodopsin chromophore and its counterion implies structural differences from bacteriorhodopsin

In wild-type proteorhodopsin (pR), titration of the chromophore's counterion Asp⁹⁷ occurs with a pK_a of 8.2±0.1. R94C mutation reduces this slightly to 7.0±0.2, irrespective of treatment with ethylguanidinium. This contrasts with the homologous archaeal protein bacteriorhodopsin (bR), where R82C mutation was previously shown to elevate the pK_a of Asp⁸⁵ by ∼5 units, while reconstitution with ethylguanidinium restores it nearly to the wild-type value of 2.5. We conclude there is much weaker electrostatic coupling between Arg⁹⁴ and Asp⁹⁷ in the unphotolyzed state of pR, in comparison to Arg⁸² and Asp⁸⁵ in bR. Therefore, while fast light-driven H⁺ release may depend on these two residues in pR as in bR, no tightly conserved pre-photolysis configuration of them is required.     

MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like Repeats

With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339–345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 °C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135–7155].Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Structure data have been deposited at PDB (1TE4) and NMR data at BMRB (5629).Olivier Julien and Isabelle Gignac - Both authors contributed equally to this work.     

A Thorough Dynamic Interpretation of Residual Dipolar Couplings in Ubiquitin

The presence of slow motions with large amplitudes, as detected by measurements based on residual dipolar couplings [Peti, W., Meiler, J., Brueschweiler, R. and Griesinger, C. (2002) J. Am. Chem. Soc., 124, 5822–5833], has stirred up much discussion in recent years. Based on ubiquitin NH residual dipolar couplings (rdcs) measured in 31 different alignment conditions, a model-free analysis of structure and dynamics [Meiler, J., Peti, W., Prompers, J., Griesinger, C. and Brueschweiler, R. (2001) J. Am. Chem. Soc., 123, 6098–6107] is presented. Starting from this broad experimental basis, rdc-based order parameters with so far unattained accuracy were determined. These rdc-based order parameters underpin the presence of new modes of motion slower than the inverse overall tumbling correlation time. Amplitudes and anisotropies of the motion were derived. The effect of structural noise on the results was proven to be negligible. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus

A bacteriochlorophyll a biosynthesis mutant of the purple photosynthetic bacterium Rhodobacter capsulatus was functionally complemented with a cosmid genomic library from Synechocystis sp. PCC 6803. The complemented R. capsulatus strain contains a defined mutation in the bchM gene that codes for Mg-protoporphyrin IX methyltransferase, the enzyme which converts Mg-protoporphyrin IX to Mg-protoporphyrin IX methylester using S-adenosyl-l-methionine as a cofactor. Since chlorophyll biosynthesis also requires the same methylation reaction, the Synechocystis genome should similarly code for a Mg-protoporphyrin IX methyltransferase. Sequence analysis of the complementing Synechocystis cosmid indicates that it contains an open reading frame exhibiting 29% sequence identity to BchM. In addition, expression of the Synechocystis gene in the R. capsulatus bchM mutant via the strong R. capsulatus puc promoter was shown to support nearly wild-type levels of bacteriochlorophyll a synthesis. To our knowledge, the Synechocystis sequence thus represents the first chlorophyll biosynthesis gene homolog of bchM. The complementing Synechocystis cosmid was also shown to code for a gene product that is a member of a highly conserved family of RNA binding proteins, the function of which in cyanobacteria remains undetermined.     

Recipe for revealing informative metabolites based on model population analysis

An important application of metabolic profiles is to discover informative metabolites/biomarkers which are predictive of a clinical outcome under investigation. Therefore, there is a need to develop statistically efficient method for screening such kind of metabolites from the candidates. The most commonly used criteria to assess variable (metabolite) importance may be the P value obtained by performing t test on each metabolite alone, without considering the influence of other variables. In this work, a new strategy, called subwindow permutation analysis (SPA) coupled with partial least squares linear discriminant analysis (PLSLDA), is developed for statistical assessment of variable importance. The main contribution of SPA is that, unlike t test, it can output a conditional P value by implicitly taking into account the synergetic effect of all the other variables. In this sense, the conditional P value could to some extent help locate a good combination of informative variables. When applied to two metabolic datasets (type 2 diabetes mellitus data and childhood overweight data), it is shown that the performance of both the unsupervised principal component analysis (PCA) and the supervised PLSLDA are greatly improved when using the informative metabolites revealed by SPA. The source codes for implementing SPA in both MATLAB and R (R package for both Linux and Windows) are freely available at: .     

Photochemistry and Photoinduced Proton-Transfer by Pharaonis Phoborhodopsin

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) ppR_K(540) ppR_KL(512) ppR_L(488) ppR_M(390) ppR_O(560) ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppR_M is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppR_M and ppR_O contain 13-cis and all-trans, respectively. ppR has the ability of lightinduced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppR_O and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.     

Use of transmissible plasmids as cloning vectors in Caulobacter crescentus

Cloning vectors for studies of Caulobacter crescentus genes should be transferable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (Su^R-Sm^R) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.     

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

西藏墨脱悬钩子属植物小志

该文在野外调查、标本采集、标本查阅与鉴定及文献考证的基础上,对西藏墨脱县产的蔷薇科悬钩子属植物进行了系统整理。结果表明:目前发现该区共有悬钩子属植物28种4变种,其中Rubus lineatus Reinw. var. glabrior Hook. f.为中国分布新记录植物,小柱悬钩子(R. columellaris Tutcher)、红毛悬钩子(R. wallichianus Wight et Arn.)、独龙悬钩子(R. taronensis C. Y. Wu ex T. T. Yu et L. T. Lu)和疏松悬钩子(R. efferatus Craib)为西藏分布新记录植物。该文还对《中国植物志》和Flora of China中该属部分学名的不恰当使用进行了探讨。     

Susceptibility of Pseudomonas aeruginosa veterinary isolates to Pbunavirus PB1-like phages

In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with ØR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.