Hiromi Nishida

Archiascomycetes : detection of a major new lineage within the Ascomycota

For phylogenetic analysis of the higher fungi, we sequenced the nuclear small subunit rRNA (18S rRNA) gene fromTaphrina populina, the type species of the genusTaphrina, andProtomyces lactucae-debilis. The molecular phylogeny inferred from these 2 sequences and 75 sequences from the DNA data bank divided the Ascomycota into three major lineages: the hemiascomycetes, the euascomycetes, and the archiascomycetes, newly described herein. The former two lineages are monophyletic, whereas the archiascomycetes, which originated first and are comprised ofTaphrina, Protomyces, Saitoella, Schizosaccharomyces, andPneumocystis, may not be monophyletic. Among the archiascomycetes, theTaphrina/Protomyces branch is monophyletic. Confirmation of the archiascomycetes as a monophyletic taxonomic class will require comparison of additional genetically defined characters.

HISTONE H3 ACETYLATED AT LYSINE 9 IN PROMOTER IS ASSOCIATED WITH LOW NUCLEOSOME DENSITY IN THE VICINITY OF TRANSCRIPTION START SITE IN HUMAN CELL

Nucleosome depletion in the promoters has been indicated in yeasts, suggesting that nucleosome depletion in promoter might be a fundamental feature of eukaryotic transcriptional regulation. We compared the relationship between histone H3 acetylation at lysine 9 (K9) in promoter, gene expression level, and nucleosome density in the vicinity of the transcription start site (TSS), in HepG2 cells (human hepatocellular liver carcinoma cells). We found that the density of nucleosome is relatively low in the close vicinity of TSS flanked by H3 K9 significantly acetylated promoter, compared with that for genes without marked H3 K9 acetylation in promoter, regardless of their transcriptional activation status. Our results imply that the relative nucleosome depletion in the vicinity of TSS is not necessarily associated with active transcription, but with histone H3 K9 acetylation in promoter.

Symbiobacterium thermophilum gen. nov., sp. nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth.

A Gram-negative and tryptophanase-positive thermophile, whose growth is dependent on co-culture with an associating Bacillus strain, had been reported and tentatively named Symbiobacterium thermophilum strain T(T). Axenic culture of strain T(T) was recently established by dialysing cultures with the supporting bacterial strains or adding their culture broth. Phylogenetic analysis of strain T(T), based on the 16S rDNA sequence, was conducted for the validation of S. thermophilum. The sequence of strain T(T) was located at the outermost position in the high-G+C Gram-positive group distinctly isolated from any other branches hitherto known. Ten sequences identical to that of strain T(T), and one sequence closely related to it, were identified for the first time from soil and compost samples. The outer membrane of strain T(T) had a three-layered structure, outside the cytoplasmic membrane, which is similar to the S-layer in the cells of members of the Bacillaceae. Chemical analysis of the cells revealed that menaquinone-6 is a major component of the quinone system. According to these results, along with several previous observations (i.e. a G+C DNA content of 65 mol% and the identification of iso-C15:0 and iso-C17:0 acids as major cellular fatty acids), the new taxon Symbiobacterium thermophilum gen. nov., sp. nov. is proposed. The type strain is S. thermophilum strain T(T) (= IAM 14863T).

The Higher Fungus Protomyces inouyei has Two Group I Introns in the 18S rRNA Gene

The nucleotide sequence of the small-subunit rRNA (18S rRNA) coding gene in the higher fungus Protomyces inouyei contains two group I introns. This is the first report of two group I introns in the 18S rRNA coding region. Based on the comparison of the two introns of Protomyces inouyei with those of the green alga Ankistrodesmus stipitatus, and the other two higher fungi Pneumocystis carinii and Ustilago maydis, the Protomyces introns are group I introns containing the highly conserved sequence elements P, Q, R, and S. Intron A of Protomyces inouyei is located in the same position as in Pneumocystis carinii while intron B shares the location with that in Ustilago maydis. The phylogenetic relationships strongly support horizontal transfer of these group I introns.

Response of the Pseudomonas host chromosomal transcriptome to carriage of the IncP-7 plasmid pCAR1

Plasmid carriage requires appropriate expression of the genes on the plasmid or host chromosome through cooperative transcriptional regulation. To clarify the impact of plasmid carriage on the host chromosome, we compared the chromosomal RNA maps of plasmid-free and plasmid-containing host strains using the incompatibility group P-7 archetype plasmid pCAR1, which is involved in carbazole degradation, and three distinct Pseudomonas strains. The possession of pCAR1 altered gene expression related to the iron acquisition systems in each host. Expression of the major siderophore pyoverdine was greater in plasmid-containing P. putida KT2440 and P. aeruginosa PAO1 than in the plasmid-free host strains, in part due to the expression of carbazole-degradative genes on pCAR1. The mexEFoprN operon encoding an efflux pump of the resistance-nodulation-cell division family was specifically upregulated by the carriage of pCAR1 in P. putida KT2440, whereas the expression of orthologous genes in the other species remained unaltered. Induction of the mexEFoprN genes increased the resistance of pCAR1-containing KT2440 to chloramphenicol compared with pCAR1-free KT2440. Our findings indicate that the possession of pCAR1 altered the growth rate of the host via the expression of genes on pCAR1 and the host chromosomes.

Evolutionary relationships of members of the genera Taphrina, Protomyces, Schizosaccharomyces, and related taxa within the archiascomycetes: Integrated analysis of genotypic and phenotypic characters

To study the phylogeny and evolution of archiascomycetes, we determined the full sequence of the nuclear 18S rRNA gene from 14Taphrina species and 2Protomyces species, and the partial sequence ofSchizosaccharomyces japonicus var.japonicus. The sequences were phylogenetically analyzed by the neighbor-joining, maximum parsimony, and maximum-likelihood methods. We also looked at their principal phenotypic characters and genotypic character. Relationships within the Ascomycota are concordant with the previously published phylogenies inferred from 18S rDNA sequence divergence and divide the archi-, hemi-and euascomycetes into distinct major lineages. All the trees show that, within the archiascomycete lineage, 11 of the 14Taphrina species and the 2Protomyces species are monophyletic. A core groups ofTaphrina andProtomyces is always monophyletic. The evidence from molecular and phenotypic characters such as cell wall sugar composition, ubiquinone, cell wall ultrastructure, and mode of conidium ontogeny, strongly suggests that ‘T’. californica CBS 374.39, ‘T’. maculans CBS 427.69 and ‘T’. farlowii CBS 376.39 should be excluded from the archiascomycete lineage. ‘Taphrina’ farlowii CBS 376.39 groups withCandida albicans in the Saccharomycetales, whereas ‘T’. californica CBS 374.39 and ‘T’. maculans CBS 427.69 have a basidiomycete affinity and group with Tremellalean members in the hymenomycete lineage.Schizosaccharomyces is monophyletic. The strictly anamorphic yeastSaitoella complicata groups with the apothecial ascomyceteNeolecta vitellina rather than theTaphrina/Protomyces branch.

Pmr, a Histone-Like Protein H1 (H-NS) Family Protein Encoded by the IncP-7 Plasmid pCAR1, Is a Key Global Regulator That Alters Host Function

Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

Phylogenetic Relationships Among Mycoplasmas Based on the Whole Genomic Information

With the rapid increase in available bacterial whole-genome information, comparison of bacteria at the whole-genome level has proven to be highly useful in microbial phylogenetic research. Here we constructed a phylogenetic tree based on 15 whole genomes of Mycoplasma and the related bacteria. First, 143 orthologous gene families that are shared by all of the 15 bacteria were selected and 143 multiple alignments were generated. Next, a concatenated multiple alignment inferred from the 143 multiple alignments was generated. A total of 43,370 amino acid sites were considered in the neighbor-joining analysis. The phylogenetic tree based on the whole-genomic information indicated that the 15 bacteria were divided into four major groups with 100% bootstrap support, i.e., the M. hyopneumoniae (Mhy) group, the M. mycoides (Mmy) group, the M. pneumoniae (Mpn) group, and the Bacillus-Phytoplasma (BP) group. In the phylogenetic tree, the Mhy group was more closely related to the Mpn group than the Mmy group. The relationships among the Mhy, Mmy, Mpn, and BP groups were supported with 100% in bootstrap analysis. The phylogenetic tree based on the whole-genome comparison is different from the 16S rRNA tree. Thirty-nine of the 143 phylogenetic trees had the same type of the topology based on the whole-genome comparison. However, we could not identify a gene family contributing or solely belonging to the topology of the 39 proteins. In this study, we showed that some proteins, such as RpoA, RpoB, RpoC, and RpoD, are not suitable for evolutionary studies on relationships among major groups of mycoplasmas. We also showed that glycolysis-related genes of Ureaplasma have a higher substitution rate than the other bacteria. The phylogenetic approaches at the whole-genome level are very important and will be essential for microbial evolutionary studies.

What Is Characteristic of Fungal Lysine Synthesis Through the α-Aminoadipate Pathway?

Abstract. Recent finding that a prokaryote synthesizes lysine through the α-aminoadipate pathway demonstrates that the lysine synthesis through the α-aminoadipate pathway is not typical of fungi. However, the fungal lysine biosynthesis is not completely the same as the prokaryotic one. We point out that α-aminoadipate reductase is a key enzyme to the evolution of fungal lysine synthesis. In addition, fungi have two different saccharopine dehydrogenases, which is also characteristic of fungi.

Multiple origins of fungal group I introns located in the same position of nuclear SSU rRNA gene

The archiascomycetous fungus Protomyces pachydermus has two group I introns within the nuclear small subunit (nSSU) rRNA gene. One of these introns has an internal open reading frame (ORF) that encodes a predicted protein of 228 amino acid residues. On the other hand, Protomyces macrosporus has two group I introns that insert at the same positions as P. pachydermus, which have no ORF. Each alignment was constructed with Protomyces group I introns located in the same position and other introns retrieved by the BLAST Search. Each phylogenetic tree based on the alignment shows that Protomyces introns are monophyletic but the relationships among fungal introns do not reflect on the fungal phylogeny. Therefore, it is suggested that two different horizontal transfers of group I introns occurred at the early stage of Protomyces species diversification.