Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.     

Roles of CfxA, CfxB, and external electron acceptors in regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase expression in Rhodobacter sphaeroides.

The Rhodobacter sphaeroides genome contains two unlinked genetic regions each encoding a series of proteins involved in CO2 fixation which include phosphoribulokinase (prkA and prkB) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcLS and rbcR) (P. L. Hallenbeck and S. Kaplan, Photosynth. Res. 19:63-71, 1988; F. R. Tabita, Microbiol. Rev. 52:155-189, 1988). We examined the effect of CO2 in the presence and absence of an alternate electron acceptor, dimethyl sulfoxide, on the expression of rbcR and rbcLS in photoheterotrophically grown R. sphaeroides. The expression of both rbcR and rbcLS was shown to depend on the CO2 concentration when succinate was used as the carbon source. It was also demonstrated that CO2 fixation is critical for photoheterotrophic growth but could be replaced by the alternative reduction of dimethyl sulfoxide to dimethyl sulfide. Dimethyl sulfoxide severely depressed both rbcR and rbcLS expression in cells grown photoheterotrophically at CO2 concentrations of 0.05% or greater. However, cells grown photoheterotrophically in the absence of exogenous CO2 but in the presence of dimethyl sulfoxide had intermediate levels of expression of rbcL and rbcR, suggesting partially independent control by limiting CO2 tension. We also present evidence for the existence of two gene products, namely, CfxA and CfxB, which are encoded by genes immediately upstream of rbcLS and rbcR, respectively. Strains were constructed which contained null mutations in cfxA and/or cfxB. Each mutation eliminated expression of the linked downstream rbc operon. Further, studies utilizing these strains demonstrated that each form of ribulose 1,5-bisphosphate carboxylase/oxygenase plays an essential role in maintaining the cellular redox balance during photoheterotrophic growth at differing CO2 concentrations.     

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

Nucleotide sequence of the Thiobacillus ferrooxidans chromosomal gene encoding mercuric reductase

The nucleotide sequence of the Thiobacillus ferrooxidans chromosomal mercuric-reductase-encoding gene (merA) has been determined. The merA gene contains 1635 bp, and shares 78.2% and 76.6% sequence homology with the transposon, Tn501, and plasmid R100 merA genes, respectively. From the sequence, a 545-amino acid (aa) polypeptide was deduced, and comparison with those of Tn501 and R100 revealed 80.6% and 80.0% homology, respectively, at the aa sequence level. Divergence among the three merA aa sequences was clustered within a specific region (aa positions 41-87). By analysis of codon usage frequency, it is speculated that the T. ferrooxidans merA gene originated from Tn501, R100, or a common ancestral gene, but not from T. ferrooxidans itself.     

Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.     

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.)

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.     

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.     

Characterization of the genomic structure and expression of the mouse Apex2 gene

We isolated a mouse cDNA encoding APEX2 protein and demonstrated that APEX2 binds to PCNA. The level of Apex2 mRNA was high in the thymus, bone marrow, spleen, and kidney in adult mice. Apex2 consists of six exons and is flanked on the 3' end by Alas2 on X chromosome 63.0. Furthermore, Apex2 is flanked on the 5' end by a novel gene with a 106-bp intergenic sequence. We disrupted Apex2 in embryonic stem cells derived from a male mouse, and a 55-kDa APEX2 protein was detected in the nuclei of Apex2(+) but not Apex2-disrupted cells. Immunoelectron microscopy revealed that APEX2 is also localized in the mitochondria of Apex2(+) cells. In serum-stimulated BALB/c 3T3 cells, the level of Apex2 mRNA was transiently increased and the level of APEX2 reached a maximum in the late S phase, thus indicating that APEX2 may participate in postreplicative base excision repair.     

A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding

The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.     

基于叶绿体基因rbcL和psbA-trnH间区探讨石杉科植物系统关系

关于石杉科Huperziaceae植物的分类,一直存在一些争议。在旧的分类体系中石杉科植物被包含在一个混合的石松科Lycopodiaceae和多谱系的石松属Lycopodium中。本文利用叶绿体rbcL基因和psbA-trnH基因间区序列探讨石杉科植物的系统位置及石杉科内部的分类关系,用最大简约法和邻接法对自测序列结合由GenBank下载的rbcL及psbA-trnH基因间区序列进行系统发育分析。结果显示,石杉科与Phylloglossum属关系较近,与石松科关系较疏远。在石杉科中热带石杉属Huperzia植物和马尾杉属Phlegmariurus植物的关系要比它们与其他石杉属植物更近。所以,我们的rbcL基因数据不支持秦仁昌关于石杉科分为石杉属和马尾杉属的分类处理。但是,因为我们的psbA-trnH序列没有包括热带种类,对石杉属植物和马尾杉属植物的关系无验证。因此需要更多的样品和序列数据进一步探讨石杉科的演化关系。     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Systematic affinities of Rhizophoraceae and Anisophylleaceae, and intergeneric relationships within Rhizophoraceae, based on chloroplast DNA, nuclear ribosomal DNA, and morphology

A cladistic analysis of sequences from the chloroplast gene rbcL was used to determine the systematic affinities of Rhizophoraceae and Anisophylleaceae. This analysis rejects close relationships of Rhizophoraceae with Celastraceae or Elaeocarpaceae, suggested previously, and identifies Erythroxylaceae as sister group within the Malpighiales, supported by several morphological and anatomical characters. Our molecular results also indicate that Anisophylleaceae are nested within Cucurbitales. Although this placement is novel, this affinity is also well supported by shared morphological characters. Tribal and generic relationships within Rhizophoraceae are evaluated with a combination of six molecular data sets (rbcL, atpB-rbcL intergenic spacer, trnL-trnF intergenic spacer, ITS1, ITS2, and 5.8S) and a morphological data set. These relationships are compared with results from previous morphological cladistic analyses. Against the background of the molecular results, we briefly discuss the evolution of morphological characters traditionally used for tribal subdivision as well as characters presumably significant for adaptation to mangrove habitats, namely, aerial stilt roots and vivipary.