L. Velasco

vnd, a gene required for early neurogenesis of Drosophila, encodes a homeodomain protein.

The development of the central nervous system in Drosophila is initiated by the segregation of neuroblasts, the neural progenitors, from the embryonic neuroectoderm. This process is guided by at least two classes of genes: the achaete‐scute complex (AS‐C) proneural genes and the neurogenic genes. It has been known for some time that loss‐of‐function mutations in the AS‐C result in neural hypoplasia and the first observed defect is failure of segregation of a fraction of neuroblasts. Loss‐of‐function mutations at the ventral nervous system defective (vnd) locus are known to lead to similar phenotypic defects in early neurogenesis. More recently, the vnd locus has been implicated in the regulation of the proneural AS‐C genes and the neurogenic genes of the Enhancer of split complex. In this paper we report the identification of a transcript associated with the vnd locus, the transcript distribution in embryogenesis, which is compatible with the nervous system mutant phenotypes described for this gene, and that the protein product is a member of the NK‐2 homeodomain family. We discuss these findings within the framework of early Drosophila neurogenesis and the known phenotypes associated with the vnd locus.

Absence of a coding region for the helper component-proteinase in the genome of cucumber vein yellowing virus, a whitefly-transmitted member of the Potyviridae

Summary.The complete nucleotide sequence of isolates of Cucumber vein yellowing virus (CVYV) has been determined. The viral genome comprises 9734 nucleotides, excluding a 3′-terminal poly(A) sequence. The genome of CVYV has a 5′-non coding and a 3′ non coding region of respectively 67 and 240 nucleotides. The RNA of CVYV encodes a single polyprotein of 3148 amino acid residues and has a deduced genome organization and motifs typical for a member of the family Potyviridae. However, CVYV is atypical because it lacks a coding sequence region for the putative helper-component as well as conserved helper-component-proteinase motifs which may account for its vector relations. All the present coding regions were compared to those from several members of the Potyviridae family. CVYV is most closely related to Sweetpotato mild mottle virus confirming its assignation to the genus Ipomovirus, despite similarities with tritimoviruses.

Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum

The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a PnosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK2.

Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).

Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum.

The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The P(norC)-lacZ fusion was not expressed in the B. japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).

One of Two hemN Genes in Bradyrhizobium japonicum Is Functional during Anaerobic Growth and in Symbiosis

Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (> or = 20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, maximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN(2), but not hemN(1), encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a Salmonella enterica serovar Typhimurium hemF hemN mutant with hemN(2) only.

Characterization of Bradyrhizobium japonicum pcaBDC genes involved in 4-hydroxybenzoate degradation.

The pca structural genes encode enzymes that participate in the conversion of protocatechuate to succinate and acetylcoenzyme A. A 3. 05-kb region of the Bradyrhizobium japonicum strain USDA110 genome has been characterized, which contains the pcaB, pcaD and pcaC genes. The predicted protein sequences of the three genes have extensive homologies with beta-carboxy-cis,cis-muconate cycloisomerase (PcaB), beta-ketodiapate enol-lactone hydrolase (PcaD), and gamma-carboxymuconolactone decarboxylase (PcaC), respectively, from Acinetobacter calcoaceticus and Pseudomonas putida. The DNA sequence revealed that the pca genes are probably arranged in a single transcriptional unit, pcaBDC, similar to that described in P. putida. A pcaB deletion mutant constructed by marker exchange mutagenesis lost the ability to use 4-hydroxybenzoate or protocatechuate as the only carbon source, demonstrating functionality of the characterized genes in catabolism of hydroxyaromatics by B. japonicum. Furthermore, 4-hydroxybenzoate and protocatechuate became toxic for the pcaB mutant, indicating that hydroxyaromatics catabolism serves both nutritional and detoxifying purposes.

Low genetic diversity among Cucumber vein yellowing virus isolates from Spain

The population structure and genetic diversity of Cucumber vein yellowing virus (CVYV) from Spain were estimated by analyses of partial nucleotide sequences of the P1-proteinase (P1-Pro), P3 protein (P3), and the coat protein (CP) coding regions. Analysis of 56 CVYV Spanish field isolates collected from 2001 to 2005 showed low genetic diversity (0.0026, 0.0013, and 0.0012 for the P1-Pro, P3, and CP regions, respectively). The ratio between nonsynonymous and synonymous substitutions was among the lowest found in a plant virus, indicating a strong negative selective pressure in the regions analyzed. Nonsynonymous nucleotide substitutions were only found within the P1-Pro regions, although these do not appear to have been selected with time. The results support the hypothesis that the Spanish CVYV population could derive from a single origin of recent introduction.

Differences of Three Ampeloviruses' Multiplication in Plant May Explain Their Incidences in Vineyards

Grapevine leafroll ampeloviruses have been recently grouped into two major clades, one for Grapevine leafroll associated virus (GLRaV) 1 and 3 and another one grouping GLRaV-4 and its variants. In order to understand biological factors mediating differential ampelovirus incidences in vineyards, quantitative real-time polymerase chain reactions were performed to assess virus populations in three grapevine varieties in which different infection status were detected: GLRaV-3 + GLRaV-4, GLRaV-3 + GLRaV-4 strain 5, and GLRaV-4 alone. Specific primers based on the RNA-dependent RNA polymerase (RdRp) domains of GLRaV-3, GLRaV-4, and GLRaV-4 strain 5 were used. Absolute and relative quantitations of the three viruses were achieved by normalization of data to the concentration of the endogenous gene actin. In spring, the populations of GLRaV-4 and GLRaV-4 strain 5 were 1.7 × 104 to 5.0 × 105 genomic RNA copies/mg of petiole tissue whereas, for GLRaV-3, values were significantly higher, ranging from 5.6 × 105 and 1.0 × 107 copies mg-1. In autumn, GLRaV-4 and GLRaV-4 strain 5 populations increased significantly, displaying values for genome copies between 4.1 × 105 and 6.3 × 106 copies mg-1, whereas GLRaV-3 populations displayed a less pronounced boost but were still significantly higher, ranging from 4.1 × 106 to 1.6 × 107 copies mg-1. To investigate whether additional viruses may interfere in the quantifications the small RNA populations, vines were analyzed by Ion Torrent high-throughput sequencing. It allowed the identification of additional viruses and viroids, including Grapevine virus A, Hop stunt viroid, Grapevine yellow speckle viroid 1, and Australian grapevine viroid. The significance of these findings is discussed.

The complete nucleotide sequence and genome organization of bean yellow disorder virus, a new member of the genus Crinivirus

Bean yellow disorder virus (BnYDV) was recently identified as the first crinivirus that infects members of the family Leguminosae [8]. The genus Crinivirus belongs to the family Closteroviridae, a family of arthropod-transmitted plant viruses that primarily infect companion and phloem parenchyma cells of their hosts [4]. All members of this virus family have large ss (+) RNA genomes ranging in size from 15 to almost 20 kb, which are encapsidated in long flexuous virions of 700–2,000 nm 9 12 nm [4]. The family Closteroviridae is divided into the genera Ampelovirus, Closterovirus and Crinivirus, which include viruses transmitted by mealybugs, aphids and whiteflies, respectively. Viruses in the genera Closterovirus and Ampelovirus have monopartite genomes, while members of the genus Crinivirus have their genomes divided into two or three separate RNAs [6]. BnYDV is transmitted by the whitefly Bemisia tabaci; the virus genome is bipartite and its sequence is unknown except for a part of the heat shock protein 70 homologue (Hsp70h) gene [8]. Here, we report the complete nucleotide sequence of a BnYDV isolate (Bn-03). Isolate Bn-03 was obtained in 2003 from a naturally infected Phaseolus vulgaris cv. Donna plant from Almeria, Spain. Double-stranded RNA was purified as described [10] and sequenced following previously reported methods: firststrand cDNA synthesis was produced using specific and random hexameric nucleotide primers [8, 9], internal cDNAs were obtained following a primer walking strategy [5] and 50-ends of both plus and minus strands were amplified with the Smart Race cDNA Amplification Kit (Clontech, Mountain View, CA, USA). Cloning and sequencing was done as described [3]. The GenBank accession numbers of the complete RNA 1 and RNA 2 sequences reported here for BnYDV are EU191904 and EU191905, respectively. The complete RNA 1 and RNA 2 sequences of the BnYDV genome consist of 8,965 and 8,530 nucleotides (nt), respectively. RNA 1 codes for a papain-like protease (L-Pro), a methyltransferase (MT) and helicase (HEL) polyprotein (ORF 1a, nt 84–6149) and an RNA-dependent RNA polymerase (RdRp) (ORF 1b, nt 6100–7665). RNA 2 contains the hallmark gene array of the family Closteroviridae coding for a Hsp70h (ORF 6, nt 1459–3129), and three diverged copies of the capsid protein: the coat protein homolog (ORF 8, nt 3288–4844), the major coat protein (ORF 10, nt 5183–5935) and the minor coat protein (ORF 11, nt 5935–7359) [6] (Fig. 1). Based on similarities to other members of the Closteroviridae, the putative protease domain [7] has two catalytic amino acids (Cys and His), while the cleavage site is predicted between Gly and Val. The conserved sequences of the BnYDV genome are most similar to those of other criniviruses (19– 79% amino acid identity). Also, a phylogenetic analysis of the most conserved gene, the RdRp, assigned BnYDV to the genus Crinivirus, well separated from viruses of the genera Ampelovirus and Closterovirus (Fig. 2). Interestingly, the closest relative of BnYDV appeared to be cucurbit yellow stunting disorder virus (CYSDV), a Bemisia tabaci-transmitted crinivirus infecting cucurbitaceous crop species. These two viruses shared (highest) amino acid sequence similarities of 73, 82, 84, and 88% in the major CP, Hsp70h, HEL, and RdRp, respectively, and were the least related in the p26/p25 (25% identity), L-Pro (34%), and P9 (38%). G. Martı́n E. Segundo I. M. Cuadrado D. Janssen (&) CIFA-Almeria, IFAPA, CICE (Junta de Andalucı́a), 04745 La Mojonera, Spain e-mail: dirkjanssen@telefonica.net