Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB¹⁵³-YMT and LamB¹⁵³-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. coli cells to bind Cd²⁺ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.     

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.     

甘蓝型油菜BnDGAT1基因表达的特异性分析

该研究从甘蓝型油菜中克隆获得了二酰甘油酰基转移酶基因（DGAT）,命名为BnDGAT1,并对该基因编码的氨基酸序列、蛋白结构域和系统进化树进行分析。结果表明:该基因编码的氨基酸序列包含二酰甘油酰基转移酶等多个功能结构域,并具有8个疏水跨膜结构区。系统进化分析表明,BnDGAT1与芥菜、拟南芥、旱金莲中DGAT1系统进化关系相对较近。利用定量PCR对BnDGAT1基因的RNA转录表达分析表明,在不同组织和角果的不同发育阶段,BnDGAT1基因的表达具有组织特异性,且在角果不同发育阶段,其RNA转录水平随着角果发育的成熟表达明显下调。     

Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12

Summary Gene lamB encodes an outer membrane protein involved in maltose and maltodextrin transport as well as phage adsorption. The active form is a trimer. We characterized 11 mutations in lamB, obtained after random insertion of a BamH1 linker and screening for stable immunodetectable mutant proteins. Six mutations resulted in the loss of the distal part of the LamB protein either by deletion (five cases) or frameshift (one case). The six corresponding proteins had all lost the ability to confer phage sensitivity and the capacity to grow on dextrins, and to yield immunnodetectable oligomers. Induction of a high level of the four longest of these proteins was toxic to the cell. Five other mutations were due to in-frame insertions. In four cases, the corresponding proteins still had the ability to yield immunodetectable oligomers, to confer phage sensitivity and the capacity to grow on dextrins and were not toxic on induction. In one case (AJC73), the mutant protein had lost the first three properties and was toxic on induction. Deletions and duplications between some of the inserts were also constructed and studied. To account for our results we present a hypothetical scheme in which trimerization would not only be needed for phage sensitivity and growth on dextrins but also for proper insertion into the outer membrane. The C-terminus of the protein, as well as other regions such as the site of mutation AJC73, would be required for the formation of stable trimers. We tentatively interpret toxicity as due to improper insertion into the outer membrane. Our results also show that it is possible to insert several amino acids (up to 11 in one case) at a number of positions in LamB without appreciably affecting its export and activities.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

结核分枝杆菌Rv3194c蛋白的亚细胞定位

【目的】Rv3194c基因编码的是结核分枝杆菌的PDZ信号蛋白,本研究探讨该蛋白的亚细胞定位,为其细胞结合蛋白的筛选奠定基础。【方法】从H37Rv基因组中扩增出编码只含有PDZ结构域的tRv3194c (Rv3194c 1–234 aa)的基因片段,在3′端加T2A和EGFP序列,一并插入真核表达载体构建出pcDNA3.1-tRv3194c-T2A-EGFP。将构建好的质粒瞬时转染L929细胞,并共感染重组痘苗病毒vTF7-3,用间接免疫荧光、流式细胞分选以及Western blotting检测融合蛋白的表达以及亚细胞定位。【结果】成功构建出真核表达载体pcDNA3.1-tRv3194c-T2A-EGFP,瞬时转染L929细胞后融合蛋白tRv3194c定位于线粒体膜上,且重组痘苗病毒vTF7-3的感染有助于靶蛋白表达水平的提高。【结论】Rv3194蛋白的PDZ结构域与线粒体外膜相关蛋白结合,为了解该蛋白在细胞内的致病机制提供重要线索。     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Site-specific mutadenesis in Enterobacter agglomerans: construction of nifB mutants and analysis of the gene's structure and function

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique, we inserted a kanamycin cassette into the cloned nifB gene, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB ^– mutants with the kanamycin cassette inserted in either orientation showed sequence of nifb. A typical ⁵⁴-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.     

Identification of a Novel mRNA-Associated Protein in Oocytes ofPleurodeles waltlandXenopus laevis

Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the completePleurodelescDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS–PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)⁺RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein–protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinantPleurodelesRAP55 fragment recognize the protein fromPleurodelesandXenopus.The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Conserved sequence motifs in the unorthodox BvgS two-component sensor protein ofBordetella pertussis

The unorthodox two-component sensor protein BvgS ofBordetella pertussis contains several interesting sequence motifs of unknown functional relevance, such as a histidine motif in its output domain, which is conserved among several unorthodox sensor proteins, a putative nucleotide binding site [Walker box type A] in its linker region, and a region in its periplasmic domain with significant homology to the TonB protein ofEscherichia coli. We investigated potential functions of these sequences by constructingB. pertussis strains that express mutant BvgS derivatives. The His₁₁₇₂ residue in the output domain was exchanged for Gln, and the Walker motif was mutated either by the replacement of Lys₆₂₅ by Arg, or of Gly₆₂₄ by Val and Lys₆₂₅ by Leu. To analyse the TonB motif, the periplasmic domain of BvgS was replaced with the corresponding domain of EvgS, anE. coli sensor that is highly homologous to BvgS but lacks the similarity with TonB. All mutations except the conservative Lys/Arg exchange in the Walker box caused the inactivation of BvgS, indicating the functional importance of the conserved motifs. The activity of the mutant proteins could be restored by complementation in trans with various separately expressed, truncated parts of BvgS. Mutations in the BvgS receiver domain could be complemented not only by a construct expressing the wild-type receiver and output domains, but also by the derivative containing the His-Gln exchange. Therefore, the histidine motif, although important for BvgS function, is not essential for complementation of BvgS mutants. The mutations in the Walker box and in the periplasmic domain could be complemented by a truncated BvgS derivative lacking the receiver and output domains. The characterization of a spontaneous revertant of the strain expressing the originally inactive EvgS/BvgS hybrid protein revealed the presence of a mutation in the BvgS linker region, conferring constitutive activity on the protein. As TonB energizes transport processes across the outer membrane ofE. coli, the strain expressing the constitutive EvgS/BvgS hybrid protein lacking the TonB motif was used in preliminary investigations of a possible direct involvement of BvgS in transport processes.     

副溶血弧菌SH112株OmpA蛋白的高效表达及免疫学特性

【目的】我们前期研究表明副溶血弧菌SH112株的OmpA蛋白在该菌的致病过程中发挥重要作用,是亚单位疫苗研制的潜在靶标抗原。本研究进一步对ompA(VPA1186)基因进行克隆表达,并研究其免疫学特性。【方法】扩增去除信号肽序列的成熟外膜蛋白OmpA的基因片段,定向克隆至表达载体,基因测序后对其编码蛋白质进行生物信息学分析。重组蛋白His-OmpA经纯化后,免疫ICR小鼠制备鼠多抗血清。Western blotting检测该蛋白的免疫原性及鼠多抗血清的特异性。动物实验验证其免疫保护率。【结果】成功表达分子量约为40.0 kDa的重组蛋白His-OmpA。制备的鼠多抗血清ELISA效价可达1∶50000以上。Westernblotting检测结果显示,该血清可与His-OmpA蛋白、总外膜蛋白和全菌蛋白发生特异性反应,说明所表达的目的蛋白保持原蛋白的免疫原性。此外,该高免血清可与其他主要血清型的副溶血弧菌发生特异性交叉反应,而与其他非副溶血弧菌菌株无交叉反应,表明该血清特异性较高,且提示OmpA蛋白可能是副溶血弧菌属的共同保护性抗原。小鼠免疫保护实验结果表明,该蛋白可提供约35%的免疫保护率。【结论】OmpA蛋白可作为诊断副溶血弧菌感染和亚单位疫苗研制的靶蛋白,为进一步开展该蛋白的功能研究提供了参考。     

Translational regulation of the Escherichia coli threonyl-tRNA synthetase gene: Structural and functional importance of the thrS operator domains

Previous work showed that E coli threonyl-tRNA synthetase (ThrRS) binds to the leader region of its own mRNA and represses its translation by blocking ribosome binding. The operator consists of four distinct domains, one of them (domain 2) sharing structural analogies with the anticodon arm of the E coli tRNA^Thr. The regulation specificity can be switched by using tRNA identity rules, suggesting that the operator could be recognized by ThrRS as a tRNA-like structure. In the present paper, we investigated the relative contribution of the four domains to the regulation process by using deletions and point mutations. This was achieved by testing the effects of the mutations on RNA conformation (by probing experiments), on ThrRS recognition (by footprinting experiments and measure of the competition with tRNA^Thr for aminoacylation), on ribosome binding and ribosome/ThrRS competition (by toeprinting experiments). It turns out that: i) the four domains are structurally and functionally independent; ii) domain 2 is essential for regulation and contains the major structural determinants for ThrRS binding; iii) domain 4 is involved in control and ThrRS recognition, but to a lesser degree than domain 2. However, the previously described analogies with the acceptor-like stem are not functionally significant. How it is recognized by ThrRS reamins to be resolved; iv) domain 1, which contains the ribosome loading site, is not involved in ThrRS recognition. The binding of ThrRS probably masks the ribosome binding site by steric hindrance and not by direct contacts. This is only achieved when ThrRS interacts with both domains 2 and 4; and v) the unpaired domain 3, which connects domains 2 and 4, is not directly involved in ThrRS recognition. It should serve as an articulation to provide an appropriate spacing between domains 2 and 4. Furthermore, it is possibly involved in ribosome binding.     

Interactions among the bHLH domains of the proteins encoded by theEnhancer of split andachaete-scute gene complexes ofDrosophila

TheEnhancer of split andachaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis inDrosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.