Functional difference between Sinorhizobium meliloti NifA and Enterobacter cloacae NifA

The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the EC nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the EC nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and EC NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix-phenotype of SmY by Sm Ni     

The Electron Transfer Flavoprotein <Emphasis Type="Italic">fixABCX</Emphasis> Gene Products from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Show a NifA-Dependent Promoter Regulation

The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric pfixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.     

<Emphasis Type="Italic">Rab11</Emphasis> is required for myoblast fusion in <Emphasis Type="Italic">Drosophila</Emphasis>

Rab11, an evolutionarily conserved, ubiquitously expressed subfamily of small monomeric Rab GTPases, has been implicated in regulating vesicular trafficking through the recycling of endosomal compartment. In order to gain an insight into the role of this gene in myogenesis during embryonic development, we have studied the expression pattern of Rab11 in mesoderm during muscle differentiation in Drosophila embryo. When dominant-negative or constitutively active Drosophila Rab11 proteins are expressed or Rab11 is reduced via double-stranded RNA in muscle precursors, they cause partial failure of myoblast fusion and show anomalies in the shape of the muscle fibres. Our results suggest that Rab11 plays no role in cell fate specification in muscle precursors but is required late in the process of myoblast fusion. This work was supported by grants from the DST (to J.K.R.) and SRF from ICMR, New Delhi (to T.B.).     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Transgenic tobacco plants containing <Emphasis Type="Italic">Bt</Emphasis> and <Emphasis Type="Italic">GNA</Emphasis> genes

A new plant expression vector (pBSbtCry1Ac-GNA) containing two insect resistant genes, a synthetic chimeric gene SbtCry1Ac encoding the insecticidal protein CrylAc and a gene GNA encoding snowdrop lectin (Galanthus nivalis agglutinin) was constructed. Transgenic tobacco plants containing these two genes were obtained through Agrobacterium-mediated transformation of tobacco leaf discs. Results from PCR detection and genomic DNA Southern blot analysis indicated that both SbtCrylAc gene and GNA gene were integrated into the genome of these plants. Results of Western blot analysis indicated that these two proteins were expressed in the analyzed plants. Bioassays of Myzus persicae and Helicoverpa assulta on detached leaves of transformed tobacco plants were carried out. The average aphid inhibition rate of these plants tested at 12 d post-infestation was 71.9 %. The average H. assulta mortality of these plants tested at 6 d post-infestation was up to 89.8 %. The kanamycin resistance of the T₁ progeny of these transgenic plants was analyzed and a typical 3:1 segregation was observed.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Extracellular production of a sphingomyelinase from <Emphasis Type="Italic">Streptomyces griseocarneus</Emphasis> using <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

The structural gene for sphingomyelinase (SMase) from Streptomyces griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl₂ which was about 50-fold more than that with 10 mM MgCl₂.     

Ectopic Expression of an <Emphasis Type="Italic">FT</Emphasis> Homolog from <Emphasis Type="Italic">Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Increase in Insecticidal Toxicity by Fusion of the <Emphasis Type="Italic">cry1Ac</Emphasis> Gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> with the Neurotoxin Gene <Emphasis Type="Italic">hwtx-I</Emphasis>

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC₅₀ (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity. LiQiu Xia and XiaoShan Long contributed equally to this work.     

Cloning and Expression of <Emphasis Type="Italic">cyt</Emphasis>2Ba7 Gene from a Soil-Isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis (Bt) cyt genes coding hemolytic and cytolytic toxins constitute a gene family, which are divided into two groups: cyt1 and cyt2. A novel cyt2 gene was detected from a soil-isolated Bt strain T301, which was highly homologous to cyt2Ba1 and finally designated cyt2Ba7. Until now, Cyt2Ba has not been expressed alone in Bt or other hosts. In this study, the cyt2Ba7 gene was cloned into the vector pQE30 and expressed as a fusion protein with 6×Histidine residues in Escherichia coli. Unlike cyt1A, cyt2Ba7 was freely expressed and formed cytoplasmic inclusions without the need for a “helper” protein. The 6×His-tagged Cyt2Ba7 was purified in one step by Ni-NTA affinity chromatography, examined cytolytic activity on Sf9 cells, and developed as an antigen to obtain the antiserum against Cyt2Ba by subcutaneous injection into rabbits. This gene was also cloned into the Bt–E. coli shuttle vector pHT3101 and expressed in Bt strain 4Q7. Immunoblotting analysis revealed that the antiserum was remarkably selective and specific to Cyt2Ba. Received: 21 December 2001 / Accepted: 28 January 2002     

Heterologous expression of leader-less <Emphasis Type="Italic">pga</Emphasis> gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis>: intracellular production of prokaryotic enzyme

Background  

Penicillin G acylase of Escherichia coli (PGA_Ec) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGA_Ec in this host and studied yeast production capacity and enzyme authenticity.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Bmrbp1</Emphasis>, the <Emphasis Type="Italic">Bombyx mori</Emphasis> homologue of the <Emphasis Type="Italic">Drosophila</Emphasis> gene <Emphasis Type="Italic">rbp1</Emphasis>

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.     

Influence of soil fertility on the rhizobial competitiveness for nodulation of <Emphasis Type="Italic">Acacia senegal</Emphasis> and <Emphasis Type="Italic">Acacia nilotica</Emphasis> provenances in nursery and field conditions

Within the framework of our study, we assessed the nodule occupancy of a mixture of various strains of rhizobia to inoculate several provenances of Acacia senegal and Acacia nilotica. The first part of the experiment was carried out under greenhouse conditions where the plants were cultivated in polyvinyl chloride tubes containing an unsterilized Sangalkam soil low in organic matter and nitrogen. The results showed that 4 and 8 months after sowing, rhizobial strains CIRADF 306 and CIRADF 300 were mainly present in nodules of A. nilotica and A. senegal, respectively. After transferring the seedlings to the more fertile soil in Bel Air field station, the molecular analysis of the nodules showed that strain CIRADF 306 was absent from the nodules of A. nilotica, whereas strain CIRADF 305 which occurred only at low nodule occupancy in the nursery, predominated in the field conditions. On the other hand, strain CIRADF 300 occurred in the majority of the nodules from the various provenances of A. senegal. These results demonstrated actual interaction between inoculated rhizobial strains, soil type and host plant genotype in terms of competitiveness, nodulation and symbiotic nitrogen fixation.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.