Identification of Tail Genes in the Temperate Phage 16-3 of Sinorhizobium meliloti 41

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.The Sinorhizobium meliloti-Medicago symbiosis is an important model for endosymbiotic nitrogen fixation. The genome sequence of S. meliloti (strain 1021) has been established (14), and the Medicago truncatula genome is under intensive investigation (3). Phage 16-3 is a temperate, double-stranded DNA phage of S. meliloti strain 41. It is by far the best-studied rhizobiophage and serves as a tool in analyses of rhizobium genetics, in the isolation of some symbiotic mutants, and in the construction of special vectors. Genetic determinants and molecular mechanisms of many aspects of the 16-3 life cycle, such as phage integration and excision (8, 26, 38), regulation of the lytic/lysogenic switch (5, 6, 9, 24, 28), immunity to superinfection (4), phage DNA packaging (15), and the role of gene h in the host range (32), have been examined in detail. Moreover, the complete 60-kb phage genome sequence (accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ500118","term_id":"111054886","term_text":"DQ500118"}}DQ500118) has been determined recently (P. P. Papp et al., unpublished results). However, little is known about the genes and structural elements involved in the interaction between the phage and its host, and furthermore, only one study of the 16-3 virion proteins has been reported (11).The initial interaction between a tailed phage and its bacterial host cell is mediated by the distal part of the phage tail, which specifically binds to the phage receptor located on the host surface. Earlier results demonstrated that phage 16-3 adsorption is connected to the strain-specific capsular polysaccharide of S. meliloti 41, the K_R5 antigen. So far, three bacterial gene clusters involved in K_R5 antigen production, including the rkp-1, rkp-2, and rkp-3 regions, have been described. rkp mutants are defective in the invasion of the host plant for symbiosis. In addition, they cannot adsorb phage 16-3, suggesting that the K_R5 antigen is required for both functions (19, 20, 30).In order to elucidate the molecular mechanism of phage 16-3 and S. meliloti 41 recognition, bacterial mutants carrying an altered phage receptor and host range phage mutants able to overcome the adsorption block have been characterized previously (32). It was shown that the RkpM protein, together with other yet uncharacterized elements, is a component of the phage receptor. With the use of rkpM mutants, host range mutations in phage gene h, which probably encodes the tail fiber protein, were identified. Interestingly, some mutations influencing phage-host recognition could not be localized in the rkpM and h genes, indicating that on both sides, additional components are important for bacteriophage-host recognition.The aim of this study was to identify additional genetic determinants involved in S. meliloti 41 and phage 16-3 recognition by characterizing new host range and receptor mutants. Furthermore, by using insertional mutagenesis, we examined a region of the phage chromosome supposed to be responsible for tail formation and identified six new genes essential for phage assembly.     

Identification of Site-Specific Recombination Genes int and xis of the Rhizobium Temperate Phage 16-3

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

噬菌体phi29末端酶gp16蛋白C端结构域在溶液中的构象分析

噬菌体phi29是一种双链DNA病毒,它的增殖和成熟过程需要将较大的子代DNA包装入一个空间极其有限的新生病毒衣壳中,这一步骤由其转运马达完成.转运马达具有三个部分:头颈连接器蛋白(the connector)、pRNA和ATP水解酶蛋白(gp16).在结构预测中发现gp16蛋白的C端结构域可能与pRNA或DNA结合,但尚未有相关文献可以证明这种相互作用,因此其结构的解析成为研究其功能的关键.本文利用大肠杆菌SUMO表达系统,对噬菌体phi29 gp16蛋白的C端结构域进行重组构建并诱导表达后,通过Ni-NTA亲和层析纯化,再用分子排阻色谱获得高纯度的目的蛋白质单体,纯度达到95%左右.最后用X射线小角散射技术(small angle X-ray scattering)对其构象进行分析,收集小角散射数据,当目的蛋白的浓度为1.3 g/L、所得到的目的蛋白的三维结构与同源蛋白FtsK的晶体结构高度拟合,且通过CRYSOL软件反推计算的曲线与实验曲线高度重合.通过原核表达、纯化及结构分析,成功获得了该结构域在溶液中的状态信息.这一研究不仅为后续关于该结构域的功能研究奠定基础,也为病毒感染的诊断和治疗提供新思路.     

Isolation and Sequencing of a Temperate Transducing Phage for Pasteurella multocida

A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae, presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNA^Leu. By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation.     

Temperate Bacteriophage ZF40 of Erwinia carotovora: Phage Particle Structure and DNA Restriction Analysis

Structural organization of the temperate bacteriophage ZF40 of Erwinia carotovora was studied. Phage ZF40 proved to be a typical member of the Myoviridae family (morphotype A1). Phage particles consist of an isometric head 58.3 nm in diameter and a contractile 86.3-nm-long tail with a complex basal plate and short tail fibers (31.5 nm). Phage tail sheath, a truncated cone in shape, is characterized by specific packaging of structural subunits. The ZF40 phage genome is 45.8 kb in size, as determined by restriction analysis, and contains DNA cohesive ends. The ZF40 phage ofErwinia carotovora is assumed to be a new species of bacteriophages specific for enterobacteria.     

Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Genetic Elements Required for Integration

Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3' ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthus genome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP x attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2. The int gene is the only Mx8 gene required in trans for attP x attB recombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP x attB recombination efficiently. The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3' end of int is replaced by host sequences. The prophage intX gene is predicted to encode an integrase with a different C terminus.     

A Study of Erwinia carotovora Phage Resistance with the Use of Temperate Bacteriophage ZF40

The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40. It was shown that, in these bacteria, the bacteriophage–cell interaction can be substantially blocked at the adsorption level. An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins. Various restriction–modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E. carotovora. The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias.     

Analysis of the Collar-Whisker Structure of Temperate Lactococcal Bacteriophage TP901-1

Proteins homologous to the protein NPS (neck passage structure) are widespread among lactococcal phages. We investigated the hypothesis that NPS is involved in the infection of phage TP901-1 by analysis of an NPS⁻ mutant. NPS was determined to form a collar-whisker complex but was shown to be nonessential for infection, phage assembly, and stability.     

Phage display-selected sequences of the heavy-chain CDR3 loop of the anti-digoxin antibody 26-10 define a high affinity binding site for position 16-substituted analogs of digoxin

The heavy-chain CDR3 region of the high affinity (K(a) = 1.3 x 10(10) M(-)1) anti-digoxin monoclonal antibody 26-10 was modified previously to shift its specificity, by substitution of tryptophan 100 by arginine, toward binding analogs of digoxin containing substitutions at position 16. To further change specificity, two 5-mer libraries of the randomly mutagenized phage-displayed 26-10 HCDR3 region (positions 94-98) were panned against digoxin-bovine serum albumin (BSA) as well as against 16-acetylgitoxin-BSA. When a mutant Fab that binds 16-substituted analogs preferentially was used as a parent sequence, clones were obtained with affinities for digoxin increased 2-4-fold, by panning on digoxin-BSA yet retaining the specificity shift. Selection on 16-acetylgitoxin-BSA, however, resulted in nine clones that bound gitoxin (16-OH) up to 150-fold higher than the wild-type 26-10, due to a consensus mutation of Ser(H95) to Gly(H95). The residues at both position H95 (serine) and position H100 (tryptophan) contact hapten in the crystal structure of the Fab 26-10-digoxin complex. Thus, by mutating hapten contact residues, it is possible to reorder the combining site of a high affinity antibody, resulting in altered specificity, yet retain or substantially increase the relative affinity for the cross-reactive ligand.     

霍乱弧菌分型噬菌体VP3蛋白的双向电泳分析

目的:噬菌体-生物分型方案具有区分霍乱弧菌潜在致病力的作用,VP3是5个霍乱弧菌分型噬菌体之一。对VP3成熟颗粒的蛋白组成进行测定和分析,以补充基因组注释信息。方法:在全基因组测序及生物信息学分析的基础上,利用双向电泳技术及质谱鉴定,对纯化的成熟VP3噬菌体的结构蛋白进行分离及鉴定。结果:双向电泳分离得到近20个蛋白点,质谱鉴定出了其中的10个,对应于4个VP3蛋白和4个霍乱弧菌蛋白。结论:VP3结构蛋白的组成和T7具有很高的相似性。与噬菌体颗粒一起被纯化分离的宿主蛋白可能在VP3的转染过程中起作用。     

The Product of the LEU-3 Cistron as a Regulatory Element for the Production of the Leucine Biosynthetic Enzymes of Neurospora

A class of intracistronic (or closely linked) partial reversions of leu-3 mutations has been found to be conditionally constitutive with respect to the synthesis of isopropylmalate isomerase (specified by the leu-2 cistron) and beta-isopropylmalate dehydrogenase (specified by the leu-1 cistron), two of the enzymes of leucine biosynthesis in Neurospora. The intermediate level of enzyme production by these leu-3(cc) mutants is independent of the obligatory inducer effector, alpha-isopropylmalate, but dependent upon the presence of the branched-chain amino acids, isoleucine, valine and leucine. The properties of leu-3+, leu-3 and leu-3(cc) in heterokaryons indicate that the transnuclear regulatory activity of the leu-3 product varies specifically as a function of available effector molecules. The information presented suggests that the leu-3 cistron is responsible not only for the production of a "positive" regulatory substance necessary for the expression of the leu-1 and leu-2 cistrons, but that it probably serves also a coordinating role in the expression of many of the genes involved in branched-chain amino acid metabolism.     

Immunological characteristics of PEP carboxylase from leaves of C3-, C4- and C3-C4 intermediate species of Alternanthera--comparison with selected C3- and C4- plants

Immunological cross-reactivity of phosphoenolpyruvate carboxylase (PEPC) in leaf extracts of C3-, C4- and C3-C4 intermediate species of Alternanthera (along with a few other C3- and C4- plants) was studied using anti-PEPC antibodies raised against PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Alternanthera, namely Amaranthaceae). Antibodies were also raised in rabbits against the purified PEPC from Zea mays (C4- monocot-Poaceae) as well as Alternanthera pungens (C4- dicot-Amaranthaceae). Monospecificity of PEPC-antiserum was confirmed by immunoprecipitation. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial immunodiffusion. Cros- reactivity of PEPC in leaf extracts from selected C3-, C4-, and C3-C4 intermediate species (including those of Alternanthera) was examined using Ouchterlony double diffusion and Western blots. Anti-PEPC antiserum raised against A. hypochondriacus enzyme showed high cross-reactivity with PEPC in leaf extracts of A. hypochondriacus or Amaranthus viridis or Alternanthera pungens (all C4 dicots), but limited cross-reactivity with that of Zea mays, Sorghum or Pennisetum (all C4 monocots). Interestingly, PEPC in leaf extracts of Alternanthera tenella, A. ficoides, Parthenium hysterophorus (C3-C4 intermediates) exhibited stronger cross-reactivity (with anti-serum raised against PEPC from Amaranthus hypochondriacus) than that of Pisum sativum, Commelina benghalensis, Altenanthera sessilis (C3 plants). Further studies on cross-reactivities of PEPC in leaf extracts of these plants with anti-PEPC antisera raised against PEPC from leaves of Zea mays or Alternanthera pungens confirmed two points--(i) PEPC of C3-C4 intermediate is distinct from C3 species and intermediate between those of C3- and C4-species; and (ii) PEPC of C4-dicots was closer to that of C3-species or C3-C4 intermediates (dicots) than to that of C4-monocots.     

ΦC31位点特异性整合酶DNA结合功能域的鉴定

DNA结合功能域的确定是阐明位点特异性重组酶整合机制的关键,而对酶DNA结合功能域进行突变研究是提高酶整合效率和整合特异性的重要方法.为了鉴定ΦC31位点特异性整合酶的DNA结合功能域,依据对ΦC31整合酶序列的生物信息学分析结果,利用PCR和克隆技术在pET22b原核表达载体上构建ΦC31整合酶重组截短突变体表达质粒,将获得的表达质粒转化入大肠杆菌BL21(DE3)菌株扩大培养并用IPTG诱导融合蛋白的表达,经镍柱纯化获得了纯度达90%以上的重组蛋白,分子量也与预期大小一致,Western印迹确定了重组蛋白的特异性.凝胶迁移滞后实验显示野生型以及截短突变体蛋白ΦC311-528、ΦC311-472、ΦC311-413能与细菌附着位点DNAattB和噬菌体附着位点DNAattP结合的条带,而截短突变体ΦC311-353、ΦC311-279、ΦC311-120观察不到相应的结合条带.6个截短突变体质粒在体内重组活性蓝白斑实验中均表现为蓝斑,显示出皆丧失体内重组活性.研究证实,ΦC31整合酶半胱氨酸富集域(第353～413位氨基酸)具有DNA结合的功能,而C末端缬氨酸富集区(第528～613位氨基酸)也与其重组活性相关.这为进一步了解ΦC31整合酶的结构与功能,最终引导其结构进化,提高其特异性和整合效率奠定了基础.