Bradyrhizobium (Arachis) sp. strain NC92 contains two nodD genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants.

The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp. strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF. nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type. Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein. nolA was required for increased expression of the nodD2-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp. strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes.     

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions

Summary By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod⁺ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material mmutant 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.Dedicated to Prof. Giorgio Semenza on the occasion of his 60th birthday     

家蚕浓核病毒 Bm DNV-3(中国株)VD1基因组结构与转录分析

为了进一步认识家蚕浓核病毒BmDNV-3(中国株)VD1基因组的结构和功能,VD1被分离、纯化、克隆到pUC119载体上,完成了基因组全序列的测定。序列分析显示VD1基因组全长为6543个核苷酸,末端拥有224个核苷酸反向重复序列(ITRs)。VD1基因组正链含有3个大的开放阅读框(ORF1-3),负链含有1个大的开放阅读框(ORF4)。比较BmDNV-3的VD1和BmDNV-2(Yamanashiisolate)的VD1基因组全序列,两者同源性为98.4%,并且有107个碱基的替代和1个碱基插入,氨基酸突变集中在VD1ORF3和VD1ORF4。Northern杂交结果显示VD1的左边正链上有1.1kb和1.5kb两个转录本,右边的负链上有一个3.3kb转录本。3′和5′-RACE结果显示1.1kb转录本开始于nt290,结束于nt1437;1.5kb转录本开始于nt1423,结束于nt2931;3.3kb转录本开始于nt6287,结束于nt2922。正链上1.5kb转录本和负链上3.3kb转录本拥有10个核苷酸的3′端的共同序列。研究结果显示该病毒基因转录与已报道的其它浓核病毒存在较大的差异性。     

Identification of Hepta- and Octo-Uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of SARS-CoV ORF 3a variants

Programmed frameshifting is one of the translational recoding mechanisms that read the genetic code in alternative ways. This process is generally programmed by signals at defined locations in a specific mRNA. In this study, we report the identification of hepta- and octo-uridine stretches as sole signals for programmed +1 and −1 ribosomal frameshifting during translation of severe acute respiratory syndrome coronavirus (SARS-CoV) ORF 3a variants. SARS-CoV ORF 3a encodes a minor structural protein of 274 amino acids. Over the course of cloning and expression of the gene, a mixed population of clones with six, seven, eight and nine T stretches located 14 nt downstream of the initiation codon was found. In vitro and in vivo expression of clones with six, seven and eight Ts, respectively, showed the detection of the full-length 3a protein. Mutagenesis studies led to the identification of the hepta- and octo-uridine stretches as slippery sequences for efficient frameshifting. Interestingly, no stimulatory elements were found in the sequences upstream or downstream of the slippage site. When the hepta- and octo-uridine stretches were used to replace the original slippery sequence of the SARS-CoV ORF 1a and 1b, efficient frameshift events were observed. Furthermore, the efficiencies of frameshifting mediated by the hepta- and octo-uridine stretches were not affected by mutations introduced into a downstream stem–loop structure that totally abolish the frameshift event mediated by the original slippery sequence of ORF 1a and 1b. Taken together, this study identifies the hepta- and octo-uridine stretches that function as sole elements for efficient +1 and −1 ribosomal frameshift events.     

Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules

The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds. We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123. Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes. Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene. Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression. In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains. The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD. An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro. Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R. l. bv. trifolii to the nonlegume Parasponia. DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined. Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.     

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.     

Localization of the T4 phage ribonucleotide reductase B1 subunit gene and the nucleotide sequence of its upstream and 5' coding regions

The nucleotide (nt) sequence in a 757-bp [corrected] segment downstream from the intron-containing T4 phage thymidylate synthase gene (td) has been determined. This region was found to contain two open reading frames (ORFs). The first ORF(ORF2) [corrected] 261 bp [corrected] in length, is 24 [corrected] nt downstream from the td gene. The second ORF(ORF3) [corrected]) is 200 bp long at 558 [corrected] nt from the td gene and extends to the end of the Eco RI fragment. The amino acid (aa) sequence (66 aa residues) deduced from the second truncated ORF shows 59% homology to the sequence of the N-terminal portion of the ribonucleotide reductase large subunit of either Escherichia coli (B1 subunit) or mouse (M1 subunit). This tentatively identifies the truncated gene to be the 5' end of the T4 phage ribonucleotide reductase subunit B1 (nrdA) gene and pinpoints its exact location on the T4 phage genomic map. Southern hybridization analysis suggests good sequence homology among the nrdA genes of various T-even phages.     

Two nifA-like genes required for expression of alternative nitrogenases by Azotobacter vinelandii.

Two nifA-like genes, designated anfA and vnfA, have been identified in Azotobacter vinelandii. The anfA gene is located upstream from the nitrogenase-3 structural gene cluster (anfHDGK) and is preceded by a sequence that is potentially part of a ntrA-dependent promoter. The product of anfA appears to be required for expression of nitrogenase-3, since cells of the anfA deletion strain CA66 were unable to synthesize this nitrogenase when derepressed in N-free, Mo- and V-deficient medium. The vnfA gene was identified after determination of the nucleotide sequence of DNA flanking the Tn5 insertion in mutant strain CA46. Two open reading frames (ORF1 and ORF2) were found located upstream from the vnfA gene, and a nifE-like ORF, preceded by a possible ntrA-dependent promoter, was found downstream from this gene. It is not known whether vnfA is expressed only under N2-fixing conditions. However, potential ntrA-dependent promoters were found immediately upstream from vnfA (within the 3' end of ORF2) and immediately downstream from ORF1. The region spanning ORF1 and ORF2 contained an A + T-rich sequence that was also found immediately upstream from the potential ntrA-dependent promoter of anfA. The product of vnfA appears to be required for the synthesis of nitrogenase-2, since cells of strain CA46 synthesized only nitrogenase-1 and -3 but not nitrogenase-2 when grown in the presence of vanadium. The product of nifA, which is required for synthesis of nitrogenase-1, is not required for synthesis of either nitrogenase-2 or nitrogenase-3. However, growth data indicate that nifA is required for a factor (or factors) necessary for maximal diazotrophic growth under Mo- and V-deficient conditions.     

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079