Host recognition and integration of filamentous phage phiRSM in the phytopathogen, Ralstonia solanacearum.

Two prophages, called varphiRSM3 and varphiRSM4, that are closely related to, but differ from, filamentous phage varphiRSM1, have been detected in strains of the Ralstonia solanacearum species complex. The prophage varphiRSM3, found in host strain MAFF730139, could be converted to infectious phage by means of PCR and transfection. The nucleotide sequence of varphiRSM3 is highly conserved relative to varphiRSM1 except for open reading frame 2 (ORF2), encoding an unknown protein, and ORF9 encoding the presumed adsorption protein that determines host range. The two host ranges differ dramatically and correlate closely with different gel electrophoresis banding patterns for cell surface fimbriae. Infections by varphiRSM1 and varphiRSM3 enhance bacterial cell aggregation and reduce the bacterial host virulence in tomato plants. Database searches in the R. solanacearum strains of known genomic sequence revealed two inovirus prophages, one designated varphiRSM4 that is homologous to varphiRSM1 and varphiRSM3, and one homologues to RSS1, in the genome of strain UW551.

Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage.

PhiRSB1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The phiRSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of phiRSB1 were demonstrated.

Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000.

PhiRSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the phiRSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5'-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the phiRSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A phiRSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these phiRSA1 genes as 5' TGTTGT-(X)13-ACAACA. The genomic sequence similarity between phiRSA1 and related phages phi52237 and phiCTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. phiRSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3' portion of the arginine tRNA(CCG) gene. In the light of the phiRSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a phiRSA1-related prophage (designated phiRSX). phiRSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. phiRSX ORFs shared very high amino acid identity with their phiRSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.

Polyploid formation between Aspergillus niger and Trichoderma viride for enhanced citric acid production from cellulose.

The first-stage heterokaryons, obtaining from intergeneric protoplast fusion between Aspergillus niger (Y-b) and Trichoderma viride (M5S51), showed slow growth and mixed morphologies on minimal medium. The fusants were classified into heterokaryon and prototrophic haploid, showing the morphology as that of A. niger. The heterokaryon strains formed conidia with the same nutritional requirements as those of the original auxotrophic mutant strains. After several subcultivations on minimal medium containing d-camphor, some heterokaryon strains formed larger two to seven nuclei/conidium as compared to one nucleus/conidium of the auxotrophic mutant and prototrophic strains, indicating that the new hybrids were generated. Interestingly, three fusant strains AT 11-2-3, AT 11-2-10, and AT 11-2-14 produce 19.2, 6.1, and 10.5 g/l citric acid, respectively, in semisolid culture containing cellulose, whereas A. niger Yang no. 2 could not use carboxymethyl cellulose as the sole carbon source for citric acid production. In addition, the average maximum beta-glucosidase and carboxymethylcellulase productions from AT 11-2-3, AT 11-2-10, and AT 11-2-14 were about 16- and 4-folds higher than those of A. niger, respectively.

New bacteriophages that infect the phytopathogen Ralstonia solanacearum.

Four kinds of bacteriophage (phiRSL, phiRSA, phiRSM and phiRSS) were isolated from Ralstonia solanacearum, a soil-borne Gram-negative bacterium that is the causative agent of bacterial wilt in many important crops. The Myovirus-type phages phiRSL1 and phiRSA1 contained dsDNA genomes of 240 kbp and 39 kbp, respectively. These phages have relatively wide host ranges and gave large clear plaques with various host strains; especially phiRSA1 was able to infect all 15 R. solanacearum strains of different races or different biovars tested in this study. Three host strains contained phiRSA1-related sequences in their genomic DNAs, suggesting a lysogenic cycle of phiRSA1. Two phages, phiRSM1 and phiRSS1, were characterized as Ff-type phages (Inovirus) based on their particle morphology, genomic ssDNA and infection cycle. However, despite their similar fibrous morphology, their genome size (9.0 kb for phiRSM1 and 6.6 kb for phiRSS1) and genome sequence were different. Strains of R. solanacearum that were sensitive to phiRSM1 were resistant to phiRSS1 and vice versa. Several R. solanacearum strains contained phiRSM1-related sequences and at least one strain produced phiRSM1 particles, indicating the lysogenic state of this phage. These phages may be useful as a tool not only for molecular biological studies of R. solanacearum pathogenicity but also for specific and efficient detection (phiRSM1 and phiRSS1) and control of harmful pathogens (phiRSL and phiRSA) in cropping ecosystems as well as growing crops.

Genomic characterization of the filamentous integrative bacteriophages {phi}RSS1 and {phi}RSM1, which infect Ralstonia solanacearum.

The genomic DNA sequences were determined for two filamentous integrative bacteriophages, phiRSS1 and phiRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of phiRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within phiRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the phiRSS1 intergenic (IG) region. The 9,004-base sequence of phiRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the phiRSM1 replication module. Genomic DNA fragments containing the phiRSM integration junctions were cloned and sequenced from phiRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5'-TGGCGGAGAGGGT-3', corresponding to the 3' end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the phiRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of phiRSS1 is within a sequence element of the IG region.

Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

ZMVHA-B1, the gene for subunit B of vacuolar H+-ATPase from the eelgrass Zostera marina L. Is able to replace vma2 in a yeast null mutant.

A vacuolar H(+)-ATPase (VHA) gene (ZMVHA-B1) was isolated from an eelgrass (Zostera marina) leaf cDNA library and was characterized to be approximately 1.4 kbp in length and to encode the B subunit protein of VHA comprising 488 amino acids. ZMVHA-B1 was highly expressed in all organs of eelgrass; the expression level was highest in the leaves. On transformation of a yeast vma2 null mutant with ZMVHA-B1, yeast cells became able to grow at pH 7.5, accompanied by the vesicular accumulation of LysoSensor green DND-189. Thus, ZMVHA-B1 expressed in yeast cells produced a functional B subunit that was efficiently incorporated into the VHA complex and eventually restored vacuolar morphology and activity. This success expedites the application of heterologous expression in yeast mutant cells to the screening of eelgrass genes involved in salt-resistance mechanisms, which are to be utilized in improving important crops.

vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2.

Cell wall materials isolated from Chlorella cells were degraded by the polysaccharide-degrading enzyme vAL-1 encoded by chlorovirus CVK2. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses of the degradation products (oligosaccharides) revealed major oligosaccharides contain unsaturated GlcA at the reducing terminus, and a side chain attached at C2 or C3 of GlcA(C4?C5), which mainly consisted of Ara, GlcNAc and Gal. The results indicated that vAL-1 is a novel polysaccharide lyase, cleaving chains of beta- or alpha-1,4-linked GlcAs. The unique structures of Chlorella cell wall were also revealed. Studies on the complicated structures of naturally occurring polysaccharides will be greatly facilitated by using vAL-1 as a tool in structural analysis.

Vp130, a chloroviral surface protein that interacts with the host Chlorella cell wall.

A protein, Vp130, that interacts with the host cell wall was isolated from Chlorovirus CVK2. From its peptide sequence, the gene for Vp130 was identified on the PBCV-1 genomic sequence as an ORF combining A140R and A145R. In Vp130, the N-terminus was somehow modified and the C-terminus was occupied by 23-26 tandem repeats of a PAPK motif. In the internal region, Vp130 contained seven repeats of 70-73 amino acids, each copy of which was separated by PAPK sequences. This protein was well conserved among NC64A viruses. A recombinant rVp130N protein formed in Escherichia coli was shown not only to bind directly to the host cell wall in vitro but also to specifically bind to the host cells, as demonstrated by fluorescence microscopy. Because externally added rVp130N competed with CVK2 to bind to host cells, Vp130 is most likely to be a host-recognizing protein on the virion.

Immediate early genes expressed in chlorovirus infections.

Twenty-three chlorovirus genes expressed in host cells as early as 5-10 min postinfection (p.i.), or immediate early, were isolated and characterized. Some showed significant homology with those for transcriptional factors and mRNA-processing proteins including TFIIB, helicases, mRNA capping enzyme, nucleolin, and bean transcription factor. Others code for (i) factors influencing translation such as aminoacyl tRNA synthetases and ribosomal protein, and (ii) unknown proteins. Enzymes involved in polysaccharide synthesis were also found. All transcripts of these genes had a poly(A) tail, which decreased in size after 20 min p.i., possibly caused by the shortening by an exonuclease. Often, due to readthrough either from an upstream ORF or into a downstream ORF, a few extra transcripts for each gene appeared after 40 min p.i., suggesting a change in promoter selection and termination accuracy at this point. A typical TATA-box and a common element 5'-ATGACAA were in the promoter region of almost all of the immediate early genes, which may be recognized by host RNA polymerase and transcription factors.

Retrotransposon-mediated restoration of Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes.

To elucidate the contribution of LINE-like retrotransposon Zepp elements to the formation and maintenance of chromosomal telomeres, newly formed minichromosomes in irradiated Chlorella vulgaris cells were isolated and structurally characterized. A minichromosome (miniV4) of approximately 700 kb in size contained a Zepp cluster taking the place of the telomeric repeats on one terminus, whereas the other end of this chromosome consisted of canonical telomeric repeats. The Zepp copies in this cluster were in a tandem array with their poly(A) tails towards the centromere. Another minichromosome Y32 ( approximately 400 kb in size) was shown to have several copies of Zepp elements on both termini. On the right arm terminus, two copies of Zepp were found in a tandem array with poly(A) tracts facing towards the chromosomal end. The poly(A) tail and the 3'-end of approximately 400 bp of the distal copy were replaced by the telomeric repeats. On the 5'-side of the proximal copy was another Zepp element in the reverse orientation. These newly formed telomeric structures are very similar to those previously found in the left arm of chromosome I and the terminus of an unidentified chromosome and support the model of Zepp-mediated restoration and maintenance of Chlorella telomeres.

Minichromosome formation in Chlorella cells irradiated with electron beams.

Three minichromosomes, miniP7, miniB7, and miniK4 of 800 kbp, 450 kbp, and 550 kbp, respectively, were obtained from Chlorella vulgaris chromosome I by electron-beam irradiation. Two of them were structurally characterized: MiniP7 was formed by the deletion of an internal 180 kbp close to the right end of chromosome I. The 180-kbp region with a small interspersed nuclear element (SINE)-like element on its left terminus was translocated to another chromosome, leaving a footprint-like structure on miniP7. MiniB7 was a hybrid of chromosome I and another chromosome, retaining the left telomere and the centromere of chromosome I. The centromeric repetitive elements served as a rearrangement point in the miniB7 formation. These examples showed the complicated mechanisms involved in the minichromosome formation. The minichromosomes thus obtained can be useful for isolating the fundamental structural elements of a chromosome. Moreover, they may serve as starting materials or a vector to generate artificial chromosomes carrying useful genes.

Chitin synthesis in chlorovirus CVK2-infected chlorella cells.

Hyaluronan synthesis in chlorovirus PBCV-1-infected Chlorella cells was previously reported (DeAngelis et al., 1997). In contrast, we report here on the detection, characterization, and expression of a gene for chitin synthase (chs) encoded by chlorovirus CVK2 isolated in Kyoto, Japan. The CVK2 chs gene encoding an open reading frame of 516 aa was expressed as early as 10 min postinfection (p.i.), peaked at 20-40 min p.i., and disappeared at 120-180 min p.i. The chitin polysaccharide began to accumulate as chitinase-sensitive, hair-like fibers on the outside of the virus-infected Chlorella cell wall by 30 min p.i. All chloroviruses without the gene for hyaluronan synthase (has) alternatively contained the chs gene, suggesting the importance of polysaccharide production in the course of virus infection. A few chloroviruses possessed both the chs and has genes and produced chitin and hyaluronan simultaneously. Polysaccharide accumulation on the algal surface may protect virus-infected algae from uptake by other organisms, such as protozoa. Since CVK2 was reported to encode two chitinases and one chitosanase, CVK2 is a very peculiar virus that encodes enzymes required for both the synthesis and the degradation of chitin materials.

Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp. strain WU-K2R.

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.

A variable region on the chlorovirus CVK2 genome contains five copies of the gene for Vp260, a viral-surface glycoprotein.

A 22.2-kb variable region near the left end of the chlorovirus CVK2 genome that was previously supposed to be expanded compared to the PBCV-1 genome was characterized. This region contains a tandem array of five gene copies for the Vp260-like protein, a viral-surface glycoprotein. The authentic 104-kDa Vp260 was found to be encoded at another site on the genome and to contain 13 internal tandem repeats of 61-65 amino acids, similar to the prominent Rickettsia surface antigen. The extra copies were also found to retain 10 of the internal repeats, despite the C-terminal deletions or extensions. These extra copies are conserved among chloroviruses isolated in various areas of Japan. By Northern blot analysis, these genes were demonstrated to be expressed late in infection. The proteins are incorporated into virions, as revealed by comparing viral structural proteins between wild-type and deletion mutants. These results indicate that extra copies of Vp260-like proteins encoded in a variable region on the genome may give variations in the surface nature of the chloroviral particles.

Recycle use of Sphingomonas sp. CDH-7 cells for continuous degradation of carbazole in the presence of MgCl2.

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.(660) 3.3) were shaken in 50 m M K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 m M MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 m M EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 m M MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 m M) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 m M) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells.

Cloning and expression of Aspergillus niger icdA gene encoding mitochondrial NADP+-specific isocitrate dehydrogenase.

The complementary DNA (cDNA) and chromosomal DNA (icdA) encoding the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Two cDNA clones (cDNA-1, 2.0 kb; cDNA-2, 1.5 kb) were obtained and sequenced, and an ORF of 1494 base pairs (bp) encoding a protein of 498 amino acids (aa) was identified in cDNA-1. The predicted amino acid sequence showed 73% and 67% sequence identities with those of the mitochondrial NADP+-ICDHs from Saccharomyces cerevisiae and pig, respectively. The sequence analysis of cDNA-1 and -2 revealed that the cDNA-2 lacks a 500-bp fragment from cDNA-1 which contains a mitochondrial targeting motif. A peroxisomal targeting motif at the C-terminus was found on the aa sequences of cDNA-1 and cDNA-2, but the cDNA-2 product seemed to be localized in the cytoplasm since the peroxisomes were not found in the mycelia of WU-2223L cultivated under the conditions of citric acid production. The expression of both cDNAs in Escherichia coli DEK2004, an isocitrate dehydrogenase-deficient mutant, revealed that both cDNAs complemented the glutamate-requiring phenotype, and that the transformants retained NADP(+)-ICDH activities. Therefore, it was clarified that both of the cDNA-1 and -2 products are fully functional. The chromosomal DNA, icdA, was cloned to correspond to cDNA-1, and its nucleotide sequence revealed that it contains seven introns. Southern hybridization using cDNA-1 and cDNA-2 indicated that there is only one copy of icdA on the chromosomes of A. niger WU-2223L. Northern hybridization analysis as for total RNA of WU-2223L revealed that two mRNAs of different sizes, 2.0 kb and 1.5 kb, were hybridized to the ORF of cDNA-1 used as a probe. Therefore, it was found that approximately 1500-nt and 2000-nt mRNAs were transcribed from only one icdA chromosomal gene in A. niger. Such a transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.

Enzymatic synthesis of alpha-arbutin by alpha-anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU-9701.

Alpha-arbutin, a useful cosmetic ingredient, was selectively synthesized by alpha-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3NaOHKCl buffer (pH 7.5) containing 1.2 M maltose at 40 degrees C, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1-O-alpha-D-glucopyranoside (alpha-arbutin) by 13C-NMR, 1H-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40 degrees C for 36 h under optimum conditions yielded 42 mM alpha-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.

Continuous degradation of dimethyl sulfoxide to sulfate ion by Hyphomicrobium denitrificans WU-K217.

With the objective of removing dimethyl sulfoxide (DMSO) contained in wastewater from semiconductor or liquid crystal display factories, biodegradation of DMSO, particularly at a low concentration, was examined. Through the screening of DMSO-degrading microorganisms, Hyphomicrobium denitrificans WU-K217 utilizing DMSO as the sole source of carbon was isolated from soil. DMSO at less than 20 mM was degraded to sulfate ion by WU-K217 with 100% molar conversion ratio based on DMSO added during 60-h cultivation at 30 degrees C under aerobic conditions. Even in the presence of 116 mM or 225 mM DMSO, WU-K217 showed growth although the amount of DMSO degraded was only 33 mM or 10 mM, respectively. Similar to the growing cells, the resting cells of WU-K217 degraded DMSO at over a wide range of temperature, 20-40 degrees C. The highest DMSO-degradation activity was obtained at 30 degrees C, and 0.64 mM (50 mg/l) DMSO was completely degraded to sulfate ion with 100% molar conversion ratio within only 15 min. Furthermore, to examine whether WU-K217 would be useful for the removal of DMSO contained in wastewater exhausted in large amounts, continuous degradation of DMSO was examined. When 0.64 mM DMSO was added to the resting cells periodically at 15-min intervals, DMSO was completely degraded to sulfate ion without any decrease of the degradation activity at least during the twelve times of DMSO addition.