Is chloroplastic class IIA aldolase a marine enzyme?

Expressed sequence tag analyses revealed that two marine Chlorophyceae green algae, Chlamydomonas sp. W80 and Chlamydomonas sp. HS5, contain genes coding for chloroplastic class IIA aldolase (fructose-1, 6-bisphosphate aldolase: FBA). These genes show robust monophyly with those of the marine Prasinophyceae algae genera Micromonas, Ostreococcus and Bathycoccus, indicating that the acquisition of this gene through horizontal gene transfer by an ancestor of the green algal lineage occurred prior to the divergence of the core chlorophytes (Chlorophyceae and Trebouxiophyceae) and the prasinophytes. The absence of this gene in some freshwater chlorophytes, such as Chlamydomonas reinhardtii, Volvox carteri, Chlorella vulgaris, Chlorella variabilis and Coccomyxa subellipsoidea, can therefore be explained by the loss of this gene somewhere in the evolutionary process. Our survey on the distribution of this gene in genomic and transcriptome databases suggests that this gene occurs almost exclusively in marine algae, with a few exceptions, and as such, we propose that chloroplastic class IIA FBA is a marine environment-adapted enzyme. This hypothesis was also experimentally tested using Chlamydomonas W80, for which we found that the transcript levels of this gene to be significantly lower under low-salt (that is, simulated terrestrial) conditions. Expression analyses of transcriptome data for two algae, Prymnesium parvum and Emiliania huxleyi, taken from the Sequence Read Archive database also indicated that the expression of this gene under terrestrial conditions (low NaCl and low sulfate) is significantly downregulated. Thus, these experimental and transcriptome data provide support for our hypothesis.

Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal environments.

Carboxydotrophic anaerobic thermophiles have been isolated from various hydrothermal environments and are considered to be important carbon monoxide (CO) scavengers or primary producers. However, the ecological factors that influence the distribution, abundance and CO-oxidizing activities of these bacteria are poorly understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus spp. in a hot spring sample and found that they constituted up to 10% of the total bacterial cells. In this study, we investigated environmental features, potential microbial CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot springs to determine environmental factors that affect CO oxidizers and to see whether Carboxydothermus spp. are common in these environments. We detected potential microbial CO-oxidation activities in samples that showed relatively high values of total organic carbon, total nitrogen, oxidation-reduction potential and soil-water content. The abundance of Carboxydothermus spp. did not correlate with the presence of potential microbial CO-oxidation activities; however, Carboxydothermus spp. were detected in a wide range of environments, suggesting that these bacteria are widely distributed in spite of the relatively low population size. This study implies that thermophilic CO oxidizers occur in a wide range of environments and oxidize CO in somewhat oxidative environments rich in organic matter.

Cloning and characterization of the gene for the iron-sulfur subunit of succinate dehydrogenase from the violet root rot fungus, Helicobasidium mompa.

The sdhB gene, encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (Sdh, EC 1.3.99.1), has been cloned from the violet root rot fungus, Helicobasidium mompa, and characterized. The promoter region contains a CCAAT box, TATA-like box, and CT-rich region. The gene is interrupted by eight introns and is predicted to encode a polypeptide of 291 amino acid residues. The putative amino acid sequence of the encoded product of sdhB gene from H. mompa shows high homology to the other known sdhB genes and is 79% identical to the Ip subunit of SdhB of Uromyces fabae. Three cysteine-rich clusters associated with the iron-sulfur centers involved in electron transport were particularly well conserved. One of these clusters contains a critical histidine residue implicated in carboxin sensitivity in the basidiomycetes. Only one copy of the gene was present in the genome of H. mompa, and reverse transcription (RT)-PCR analysis of mRNA expression showed that the sdhB gene was transcribed in potato dextrose broth.

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix.

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25 degrees C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system f or insertional mutagenesis in R necatrix and provide a simple and reliable method for genetic manipulation.

Ambient pH signaling regulates expression of the serine protease gene (spr1) in pine wilt nematode-trapping fungus, Monacrosporium megalosporum.

We have cloned and characterized spr1, a putative serine protease gene, from a nematode-trapping fungus, Monacrosporium megalosporum. The gene was present as a single copy in the genome. The predicted protein sequence of spr1 is homologous to the putative cuticle-degrading serine proteases PII and Azo1 from the nematode-trapping fungus, Arthrobotrys oligospora. In the 5' untranslated region near the initiation codon, consensus sequences to an AreA binding site, a well-known mediator of nitrogen metabolite repression in the fungus Aspergillus nidulans, a CreA binding site, a carbon response regulator in A. nidulans, and a PacC binding site, a transcription factor that responds to ambient pH signals in A. nidulans were found. However, spr1 was not regulated by carbon or nitrogen source, and exogenous protein did not induce expression of spr1. The transcription of the spr1 gene of this fungus was significantly affected by ambient pH. Based on RT-PCR, the product of the spr1 gene was not transcribed at pH 4, whereas under alkaline conditions such as pH 8 and 9, the spr1 gene was transcribed well. These results indicate that the spr1 gene is controlled only by a PacC homologue. Moreover, the expression profile of the spr1 gene corresponded with the pH-dependent physiology of this fungus.

Physiology and molecular characteristics of a pine wilt nematode-trapping fungus, Monacrosporium megalosporum.

We isolated the nematode-trapping fungus Monacrosporium megalosporum from nature and examined its morphology, physiology and molecular characteristics. The nematode-trapping device of this fungus is a three-dimensional network. This fungus captures the pine wilt nematode (Bursaphelenchus xylophilus), but not a non-phytopathogenic nematode that is morphologically similar to B. xylophilus. The phylogenic relationship of the nucleotide sequence of the rDNA ITS region was close to those of M. thaumasium and Geniculifera eudermata, which also have nematode-trapping devices that are three-dimensional networks. Acidic pH inhibited both the liberation and regeneration of protoplasts. Moreover, cytoplasmic granulation of protoplasts was found below pH 6.0. Mycelial growth on agar media was also inhibited below pH 4, but not at pH 9. These results strongly suggest that the activity of this fungus is inhibited by acid rain in the field. Therefore, development of pine wilt disease might be a secondary effect of acid rain.

Heterologous diploid nuclei in the violet root rot fungus, Helicobasidium mompa.

Allelic genes hga1-1 and hga1-2, which encode G protein alpha subunit in the violet root rot fungus, Helicobasidium mompa, were sequenced and characterized. Restriction fragment polymorphism (RFLP) analysis determined that the gene is present as a single locus in the single basidiospore isolates, while strain V169 possessed both alleles of this gene. Therefore, although basidiospore isolates are dikaryon, they are homokaryotic. Field-isolated strain V169, the putative parent strain, is a dikaryotic heterokaryon. Allelic genes hga1-1 and hga1-2 segregated in almost a 1:3 ratio among single basidiospore isolates from the same fruiting body. Moreover, the copy number of hga1-1 was found to be less than that of hga1-2 in the V169 strain. These results suggest that one of the nuclei in the V169 parent strain is homozygous diploid and the other heterozygous diploid. This parent strain produced four homokaryotic and dikaryon basidiospores on each basidium.

Telomeric fingerprinting of the violet root rot fungus, Helicobasidium mompa: a useful tool for karyotype estimation.

We hybridized the telomere-associated DNA sequence pTel46 isolated from Coprinus cinereus with Helicobasidium mompa genomic DNA. The hybridized fragments were more sensitive to Bal31 nuclease than those that were not hybridized, suggesting that they were located at the ends of chromosomes in H. mompa. The hybridization profile can be used to estimate chromosome number, since the number of chromosomes in a single basidiospore isolate is about half that in putative parent strains. Thus, single basidiospore and field isolates might be homokaryons and heterokaryons respectively. We found telomere-linked restriction fragment length polymorphisms (RFLPs) in strains of H. mompa isolated from field and individual basidiosporcs. Thus, this marker appears to be an excellent tool with which to reveal the considerable polymorphism of H. mompa and to identify strains. The RFLP was not found among several strains of the same mycelial compatibility group (MCG) isolated from the same field, suggesting that strains belonging to an MCG group are identical.

Telomeric fingerprinting of the white root rot fungus, Rosellinia necatrix: a useful tool for strain identification.

The telomere associated DNA sequence pTel46, which was isolated from Coprinus cinereus, was hybridized with Rosellinia necatrix genomic DNA. The DNA fragments hybridized with pTel46 were more sensitive to Bal31 nuclease. This result suggests that the DNA fragments hybridized with pTel46 were located at the end of chromosomes in R. necatrix. Telomere-linked restriction fragment length polymorphism (RFLP) was found in strains of R. necatrix isolated from various field and single ascospores. Thus, this marker appears to be an excellent tool to show the great polymorphism of R. necatrix. However, RFLP could not be found among several field isolated strains belonging to the same mycelial compatibility group (MCG) isolated in the same field. Therefore the strains belonging to the same MCG might be the same strain that could be anastomosed with each other without cell death except for strain W718 carrying a double-stranded RNA (dsRNA) virus. Therefore the RFLP corresponded to a MCG group, and none of the strains belonging to the same MCG group showed different RFLP in R. necatrix. Moreover, the presence of a kind of dsRNA virus might imply anastomosis between compatible strains.