Genetic Analysis of the System that Reduces Biotin-d-Sulfoxide in Escherichia coli

Four genes of Escherichia coli whose products are needed to reduce biotin-d-sulfoxide to biotin have been mapped: bisA next to chlA, bisB next to chlE, bisC linked to xyl, and bisD next to chlG. A defective lambda transducing phage, lambdadbis5, which carries all the bacterial genes between the lambda attachment site and chlE, was isolated and shown to have lost the phage genes from int through Q.     

Identification of an iron uptake system specific for coprogen and rhodotorulic acid in Escherichia coli K12

With the lac operon fusion technique, mutants were isolated in two genes that specify two outer membrane proteins designated FhuE (76 K) and Fiu (83 K). The synthesis of both proteins was increased under low iron growth conditions. The FhuE-protein was shown to be necessary for iron uptake via coprogen, an iron chelator produced by certain fungi, e.g. Neurospora crassa. In addition to fhueE the genes fhuCDB, tonB and exbB were necessary for iron coprogen uptake. The gene fhuE was mapped between kdp and gltA near 16 min on the genetic map of E. coli K12, while gene fiu was mapped near 18 min between chlA and chlE. Nor iron transport system could be assigned as yet to the Fiu protein.     

Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli.

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.     

Synthesis of nitrate reductase components in chlorate-resistant mutants of Escherichia coli.

Specific antibody to purified nitrate reductase from Escherichia coli was used to identify enzyme components present in mutants which lack functional nitrate reductase. chlA and B mutants contained all three subunits present in the wild-type enzyme. Different peptides with a broad range of molecular weights could be precipitated from chlCmutants, and chlE mutants contained either slightly degraded enzyme subunits or no precipitable protein. No mutants produced significant amounts of cytoplasmic enzyme. The chlA and B loci are suggested to function in the synthesis and attachment of a molybdenum-containing factor. The chlC locus is suggested to be the structural gene for nitrate reductase subunit A and chlE is suggested to be involved in the synthesis of the cytochrome b1 apoprotein.     

Involvement of chlA, E, M, and N loci in Escherichia coli molybdopterin biosynthesis.

All molybdenum enzymes except nitrogenase contain a common molybdenum cofactor, whose organic moiety is a novel pterin called molybdopterin (MPT). To assist in elucidating the biosynthetic pathway of MPT, two MPT-deficient mutants of Escherichia coli K-12 were isolated. They lacked activities of the molybdenum enzymes nitrate reductase and formate dehydrogenase, did not reconstitute apo nitrate reductase from a Neurospora crassa nit-1 strain, and did not yield form A, a derivative of MPT. By P1 mapping, these two mutations mapped to chlA and chlE, loci previously postulated but never definitely shown to be involved in MPT biosynthesis. The two new mutations are in different genetic complementation groups from previously isolated chlA and chlE mutations and have been designated as chlM and chlN (closely linked to chlA and chlE, respectively). The reported presence of Mo cofactor activity in the chlA1 strain is shown to be due to in vitro synthesis of MPT through complementation between a trypsin-sensitive macromolecule from the chlA1 strain and a low-molecular-weight compound from the nit-l strain.     

Alterations in the Cytoplasmic Membrane Proteins of Various Chlorate-Resistant Mutants of Escherichia coli

Chlorate-resistant mutants corresponding to each known genetic locus (chlA, chlB, chlC, chlD, chlE) were isolated from Escherichia coli K-12. All these mutants showed decreased amounts of membrane-bound nitrate reductase, cytochrome b, and formic dehydrogenase, but all had normal succinic dehydrogenase activity. Proteins from the cytoplasmic membranes of these mutants were compared to those of the wild type-on polyacrylamide gels. The addition of nitrate to wild-type anaerobic cultures caused increased formation of three membrane proteins. These same proteins, along with one other, were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase, although this protein was present in some mutants lacking nitrate reductase activity. None of the others has been identified.     

Synthesis and degradation of nitrate reductase in Escherichia coli.

The biosynthesis, insertion, and in vivo stability of nitrate reductase were examined by following the amount of labeled enzyme present in both membranes and cytoplasm at varying times after a short pulse of radioactive sulfate. Nitrate reductase levels were measured by autoradiography of immunoprecipitated material after fractionation on sodium dodecyl sulfate-polyacrylamide gels. These experiments demonstrated that subunits A and B were synthesized in the cytoplasm and subsequently inserted into membranes. The insertion of these subunits was dependent upon the synthesis of another protein, and the rate of synthesis of this protein determined the rate of insertion of subunits A and B. The nitrate reductase produced by the chlA mutant was inserted into membranes in the normal fashion, whereas the nitrate reductase produced by the chlC and chlE mutants was poorly incorporated. The nitrate reductase in the wild type was completely stable in vivo under inducing or noninducing conditions, whereas in the chlC and chlE mutants nitrate reductase was degraded extensively in both the cytoplasm and membranes, even under inducing conditions. Under similar conditions, nitrate reductase was stable in the chlA mutant.     

Reconstitution of Nitrate Reductase Activity and Formation of Membrane Particles from Cytoplasmic Extracts of Chlorate-Resistant Mutants of Escherichia coli

The reconstitution of nitrate reductase activity in mixtures of cytoplasmic fractions from the chlorate-resistant mutants chlA, B, C, and E which are lacking this activity was investigated, and the membrane-like particulate material which formed during this reconstitution was analyzed by polyacrylamide gel electrophoresis. When chlA and chlB extracts are incubated together, the cytoplasmic membrane proteins present in the particles which are formed are contributed by both mutants, and the proteins are essentially the same as the proteins in the cytoplasmic membrane fractions of the two mutants. Identical amounts of protein become particulate when cytoplasmic extracts of any of the mutant strains or wild-type strains are incubated at 32 C either singly or in mixtures, and the formation of particulate material does not appear to be a consequence of nitrate reductase reconstitution. Experiments with wild-type strains indicate that the membrane proteins in the cytoplasmic extract are derived from the cytoplasmic membrane during cell breakage. Reconstitution experiments involving various combinations of preincubated and unincubated extracts of the mutants have allowed a preliminary identification of three types of components which are necessary for the formation of active nitrate reductase: (i) a soluble factor present only in extracts from induced chlB; (ii) a different soluble factor which is missing in chlB but is present in extracts from wild-type, chlA, chlC, and chlE; and (iii) a complex including the nitrate reductase protein which is inactivated by preincubation of the mutant extracts.     

Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli.

Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.     

植物TIR-NB-LRR类型抗病基因各结构域的研究进展

植物抗病反应是一个多基因调控的复杂过程,在这个过程中R基因发挥了非常重要的作用。根据其氨基酸基序组成以及跨膜结构域的不同,R基因可以分为多种类型,其中NBS-LRR类型是植物基因组中最大的基因家族之一。TIR-NB-LRR类型的抗病基因又是NB-LRR类型中的一大类,也是目前抗病基因研究的热点。该文总结了TIR-NB-LRR类型抗病基因各个结构域的功能和相关的研究进展。相关研究表明,TIR结构域主要通过自身或异源的二聚体化介导抗性信号的转导,但也有部分研究表明,该结构域可能参与病原菌的特异性识别。NBS结构域常被认为具有"分子开关"的功能,它可以通过结合ADP或ATP来调节植物抗病蛋白的构象变化,从而调节下游抗病信号的传导。LRR结构域在植物与病原菌互作的过程中可以通过与病原菌的无毒蛋白直接或间接互作来特异识别病原菌。也有研究发现,LRR结构域具有调节信号传导的功能。这些信息将为研究植物抗病机理提供理论依据,也为将来通过基因编辑技术对作物进行定向抗病育种提供思路。     

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants.     

Localization of germin genes and their products in developing wheat coleoptiles

Germination is a process which characterized with nescient synthesis of genes. Among the genes synthesized during the germination of wheat embryos, germin genes, proteins and their enzymatic activity were defined. Germin is a water soluble homopentameric glycoprotein which is remarkable resistant to degradation by a broad range of proteases including pepsin. Germin proteins found to have strong oxalate oxidase activity which produces hydrogen peroxide by degrading oxalic acid. The current study, aimed to localize the germin genes, proteins and enzymatic activities in developing coleoptiles which is a rapidly growing protective tissue of leaf primordium and shoot apex. Non-radioactively labeled germin riboprobes were employed to localize germin mRNAs in situ. FITC (Fluorescein isothiocyanate) and alkaline phosphatase linked anti-germin antibodies were used to localize germin proteins under the fluorescence and light microscopy and finally germin enzymatic activity was localized by using appropriate enzyme assay. The results revealed that in coleoptiles germin genes, proteins and their enzymatic activity were predominantly associated with the cells of epidermis and vascular bundle sheath cells.     

Evolution of antiparallel two-domain membrane proteins: tracing multiple gene duplication events in the DUF606 family

X-ray crystallography has revealed that many integral membrane proteins consist of two domains with a similar fold but opposite (antiparallel) orientation in the membrane. The proteins are believed to have evolved by gene duplication and gene fusion events from a dual topology ancestral membrane protein, that adapted both orientations in the membrane and formed antiparallel homodimers. Here, we present a detailed analysis of the DUF606 family of bacterial membrane proteins that contains the entire collection of intermediate states of such an evolutionary pathway: single genes that would code for dual topology homodimeric proteins, paired genes coding for homologous proteins with a fixed but opposite orientation in the membrane that would form heterodimers, and fused genes that encode antiparallel two-domain fusion proteins. Two types of paired genes can be discriminated corresponding to the order in which the genes coding for the two oppositely oriented proteins occur in the operon. On the protein level, the heterodimers resulting from the two types of gene pairs are indistinguishable. In contrast, two types of fused genes corresponding to the two possible orders in which the oppositely oriented domains are present in the encoded proteins, do result in discernible types of proteins. The large number of genetic and protein states in the DUF606 family allowed for a detailed phylogenic analysis that revealed a total of nine independent duplication events in the DUF606 family, five of which resulted in paired genes, and four resulted in fused genes. Noticeably, there was no evidence for a sequential mechanism in which fusions evolve from a pair of genes. Rather, an evolutionary mechanism is proposed by which antiparallel two-domain proteins are the direct result of a gene duplication event. Combining the phylogeny of proteins and hosting microorganisms allowed for a reconstruction of the evolutionary pathway.     

Construction of a series of ompF-ompC chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of the translational products.

OmpF and OmpC are major outer membrane proteins. Although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine the difference, a method was developed to construct a series of ompF-ompC chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The genomic structures of these chimeric genes were determined by restriction endonuclease analysis and nucleotide sequence determination. In almost all cases, recombination took place between the corresponding homologous regions of the ompF and ompC genes. Many of the chimeric genes produced proteins that migrated to various positions between the OmpF and OmpC proteins on polyacrylamide gel. On the basis of the results, a domain contributing to the mobility difference the OmpF and OmpC proteins was identified. Some chimeric genes did not accumulate outer membrane proteins, despite the fact that the fusion of the ompF and ompC genes was in frame. Bacterial cells possessing the chimeric proteins were also tested as to their sensitivity to phages which require either OmpF or OmpC as a receptor component. The chimeric proteins were either of the OmpF or OmpC type with respect to receptor activity. Based on the observations, the roles of submolecular domains in the structure, function, and biogenesis of the OmpF and OmpC proteins are discussed.     

Molecular characterization of the immune system: emergence of proteins,processes, and domains

Many genes and proteins are required to carry out the processes of innate and adaptive immunity. For many studies, including systems biology, it is necessary to have a clear and comprehensive definition of the immune system, including the genes and proteins that take part in immunological processes. We have identified and cataloged a large portion of the human immunology-related genes, which we call the essential immunome. The 847 identified genes and proteins were annotated, and their chromosomal localizations were compared to the mouse genome. Relation to disease was also taken into account. We identified numerous pseudogenes, many of which are expressed, and found two putative new genes. We also carried out an evolutionary analysis of immune processes based on gene orthologs to gain an overview of the evolutionary past and molecular present of the human immune system. A list of genes and proteins were compiled. A comprehensive characterization of the member genes and proteins, including the corresponding pseudogenes is presented. Immunome genes were found to have three types of emergence in independent studies of their ontologies, domains, and functions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli.

Escherichia coli used tetrahydrothiophene 1-oxide (THTO) as an electron acceptor for anaerobic growth with glycerol as a carbon source; the THTO was reduced to tetrahydrothiophene. Cell extracts also reduced THTO to tetrahydrothiophene in the presence of a variety of electron donors. Chlorate-resistant (chl) mutants (chlA, chlB, chlD, and chlE) were unable to grow with THTO as the electron acceptor. However, growth and THTO reduction by the chlD mutant were restored by high concentrations of molybdate. Similarly, mutants of E. coli that are blocked in the menaquinone (vitamin K2) biosynthetic pathway, i.e., menB, menC, and menD mutants, did not grow with THTO as an electron acceptor. Growth and THTO reduction were restored in these mutants by the presence of appropriate intermediates of the vitamin K biosynthetic pathway.     

Secreted proteins of Uromyces fabae: similarities and stage specificity

Uromyces fabae on Vicia faba is a model system for obligate biotrophic interactions. Searching for potential effector proteins we investigated the haustorial secretome of U. fabae (biotrophic stage) and compared it with the secretome of in vitro grown infection structures, which represent the pre-biotrophic stage. Using the yeast signal sequence trap method we identified 62 genes encoding proteins secreted from haustoria and 42 genes encoding proteins secreted from in vitro grown infection structures. Four of these genes were identical in both libraries, giving a total of 100 genes coding for secreted proteins. This finding indicates a strong stage-specific regulation of protein secretion. Similarity with previously identified proteins was found for 39 of the sequences analysed, 28 of which showed similarity to proteins identified among members of the order Uredinales only. This might be taken as an indication for possible roles in virulence and host specificity unique to the Uredinales.     

Localization of functional domains in E. coli K-12 outer membrane porins.

The genes ompC and phoE of Escherichia coli K-12 encode outer membrane pore proteins that are very homologous. To study the structure-function relationship of these proteins, we have constructed a series of ompC-phoE hybrid genes in which the DNA encoding part of one protein is replaced by the corresponding part of the other gene. These hybrid genes were easily obtained by using in vivo recombination. The fusion sites in the hybrid genes were localized by restriction enzyme mapping. The hybrid gene products were normally expressed and they were characterized with respect to functions and properties in which the native OmpC and PhoE proteins differ, such as pore characteristics, the receptor activity for phages and the binding of specific antibodies. Three regions within the N-terminal 130 amino acids were localized which determine pore characteristics and a segment between residues 75 and 110 contains amino acids which determine specificity for PhoE phages. A major cell surface-exposed region is located between residues 142 and 267. This region contains residues which are required for the binding of monoclonal antibodies directed against the cell surface-exposed part of PhoE and residues which determine specificity for OmpC phages.