Mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae is organized to form mitochondrial nucleoids (mt-nucleoids) by association with specific proteins. The sensitivity of DNA-protein complexes to nuclease digestion is a useful means for examining DNA packaging and organization. However, the mt-nucleoids isolated from wild-type cells of S. cerevisiae demonstrated a significant amount of endogenous nuclease activity. In order to minimize the nuclease activity associated with the isolated mt-nucleoids, we isolated the mt-nucleoids from a mutant strain that lacked the mitochondrial nuclease, Nuc1p. In this manner, we succeeded in isolating mt-nucleoids that showed a low level of the nuclease activity. Micrococcal nuclease treatment of these mt-nucleoids led to the continuous digestion of mtDNA in the presence of Ca2+ ions. MtDNA in the mt-nucleoids also showed the continuous digestion pattern when treated with DNase II. These results suggest that mtDNA in the mt-nucleoids is protected from nuclease digestion by association with proteins, but the organization of the mtDNA-protein complexes is different from that of nuclear chromatin, in which the unit of DNA packaging is regularly repeated.
A new thermophilic spore-forming strain KG8T was isolated from the mud of Taslidere hot spring in Batman. Strain KG8T was aerobe, Gram-positive, rod-shaped, motile, occurring in pairs or filamentous. Growth was observed from 35-65ºC (optimum 55ºC) and at pH 5.5-9.5 (optimum pH 7.5). It was capable of utilizing starch, growth was observed until 3% NaCl (w/v) and it was positive for nitrate reduction. On the basis of 16S rRNA gene sequence similarity, strain KG8T was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone—menaquinone-7; major fatty acid—iso-C15:0 and iso-C17:0) supported the affiliation of strain KG8T to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain KG8T. Based on these results we propose assigning a novel subspecies of Anoxybacillus kamchatkensis, to be named Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov. with the type strain KG8T (DSM 18475T=CIP 109280T).
The taxonomic position of one soil isolate 13-12(50)T was clarified by a polyphasic study. The organism showed a combination of chemotaxonomic and morphological properties typical of the genus Actinomadura. It formed a distinct phyletic line in the Actinomadura 16S rRNA gene tree and was closely associated with Actinomadura mexicana (sequence similarity 99.5%), Actinomadura glauciflava, Actinomadura citrea (sequence similarity 99.4%) and Actinomadura madurae (sequence similarity 99.2%). The result of DNA-DNA hybridizations between 13-12(50)T and Actinomadura mexicana was 49.9%. On the basis of the phenotypic, chemotaxonomic and genotypic properties, the isolate was differentiated from its closest phylogenetic relatives. It is proposed that the organism be classified as a novel species of the genus Actinomadura. The name proposed for the new taxa are Actinomadura maheshkhaliensis sp. nov. [(13-12(50)T=JCM 13934T =MTCC 8055T].
To achieve high expression of glycoside hydrolase family 45 endoglucanase (RsSym45EG1) from a symbiotic protist of the termite Reticulitermes speratus, synthetic sequence RsSym45eg1-co, in which the codon usage was adjusted to that of the highly-expressed tef1 gene encoding translation elongation factor 1α, was prepared and introduced into A. oryzae. The transcript level of RsSym45eg1-co was 1.8-fold higher than that of RsSym45eg1. In cells harboring RsSym45eg1, but not RsSym45eg1-co, truncated transcripts in which the coding region was prematurely terminated and followed by a poly A chain were found. The production of endoglucanase in the culture supernatant was improved by codon optimization. Truncated transcripts were also found when cellobiohydrolase and β-glucosidase from R. speratus symbionts were expressed, and the transcript level of the former was increased by codon-optimization. Our findings suggest that premature polyadenylation frequently occurs in heterologous protein expression in A. oryzae, which might result in the poor yield of expressed proteins.
During a survey of yeast strains having high conversion efficiency to ethanol from cellobiose, ‘Ogataea pini’ ATCC 28781 and ‘Pichia pini’ NBRC 1794 were found to be distinct from any known species and from each other by a BLAST homology search using the D1/D2 LSU rRNA gene sequences. The D1/D2 phylogeny showed that ‘O. pini’ ATCC 28781 and ‘P. pini’ NBRC 1794 belonged to the Ogataea cluster, whereas a comparison of the ITS 1 and 2 regions sequences showed that the ATCC and NBRC strains each formed a species distinct from O. ganodermae, O. pini, O. henricii, and P. zsoltii, based on the D1/D2 sequence divergence. The ATCC and NBRC strains formed two to four hat-shaped ascospores and two to four, or more ones per deliquescent ascus, respectively, were negative for DBB and urease reactions, assimilated methanol slowly and nitrate not at all, and had the major ubiquinone system Q-7. These characteristics coincided basically with the definition of Ogataea proposed by Yamada et al. in 1994, excluding the number of ascospores. On the other hand, the ATCC and NBRC strains differed not only from each other but from relatives in various phenotypic characteristics. These differences suggest that two new yeasts of Ogataea be described as novel. The new species and their type strains are as follows: O. neopini ATCC 28781T; and O. corticis NBRC 1794T. In addition, the emendation of the genus Ogataea is made; besides, we propose the transfer of P. zsoltii, P. dorogensis, and P. trehaloabstinens, which were placed in the Ogataea cluster based on the D1/D2 sequence analysis, to the genus Ogataea as O. zsoltii comb. nov., O. dorogensis comb. nov., and O. trehaloabstinens comb. nov.
This paper outlines the construction of a novel vector system comprising interchangeable terminators, as well as a multiple cloning site (MCS), to facilitate the transformation of the fungal plant pathogen Botrytis cinerea. Previous molecular studies on B. cinerea have relied upon the pLOB1 based vector system (controlled by the Aspergillus nidulans oliC promoter and a region reported to be the B. cinerea tubA terminator). Investigations, however, have revealed that, rather than the genuine B. cinerea tubA terminator, the pLOB1 terminator fragment is from another gene locus within the genome. Because previous studies have found that terminators aide in transcript stability, the main aims of this study were to develop and evaluate both vector systems, pOT (controlled by the A. nidulans oliC promoter and A. nidulans trpC terminator) and pLOB, with a range of exogenous genes, including enhanced green fluorescent protein (eGFP), monomeric red fluorescent protein (mRFP), luciferase (LUC) and β-glucuronidase (GUS). Our investigations demonstrate that pLOB and pOT based vectors are capable of expressing all four reporter genes and may be applied to future molecular studies on B. cinerea and other related ascomycetes. Additionally, this is the first reported expression of mRFP and LUC in B. cinerea.
Two yeast strains isolated from galleries of ambrosia beetles in Japan and maintained in NITE Biological Resource Center (NBRC) as Pichia pini were found to represent a hitherto undescribed species. This species shows close relationship to Pichia dorogensis by the sequence analysis of the D1/D2 domain of 26S rDNA but is clearly differentiated from it by a DNA-DNA reassociation experiment. It is described as Ogataea paradorogensis sp. nov. The vegetative cells and asci of this species are surrounded with distinct capsules like P. dorogensis. One to four hat-shaped ascospores, which tend to be liberated from the asci at maturation, are formed in the ascus.
A novel psychro-tolerant bacterium, Curtobacterium luteum, secreting an extracellular protease was isolated from the soil of Gangotri glacier, Western Himalaya. Maximum enzyme production was achieved when the strain was grown in a pH-neutral medium containing skim milk at 15ºC over 120 h. The metal ions such as Zn2+ and Cr2+ enhanced enzyme production. The specific activity of purified enzyme was 8,090 units/mg after 34.1-fold purification. The 115 kDa enzyme was a metalloprotease (activity inhibited by EDTA and EGTA) and showed maximum activity at 20ºC and pH 7. The enzyme was active over a broad pH range and retained 84% of its original activity between pH 6 and 8. There was no loss in enzyme activity when exposed for 3 h at 4–20ºC. However, the enzyme lost 65% of activity at 30ºC, and was almost inactivated at 50ºC, but was resistant to repeated freezing and thawing. The enzyme activity was stimulated by manganese ions; however, it was inactivated by copper ions.
LZ-8, a fungal immunomodulatory protein found in Ganoderma lucidium, has been proposed to have therapeutic effects on cancer and autoimmune diseases. To develop an efficient and facile expression system supplying of rLZ-8 of high purity and stable activity for further study and applications, a full-length cDNA of lz8 gene was cloned into the pPIC9K to construct a yeast expression vector pPIC9K-lz8, then transformed to Pichia pastoris strain GS115. The molecular weight of the rLZ-8 was about 17 kDa. The yield of rLZ-8 reached up to 270 mg L-1 at optimal culture conditions. In vitro, the rLZ-8 was capable of hemagglutinating mouse red blood cells, but no such activity was observed toward human red blood cells, which were similar to the native LZ-8. The mouse splenocyte proliferation activity of rLZ-8 expressed in P. pastoris was much higher than the rLZ-8 expressed in E. coli. All these results suggested that the active rLZ-8 protein can be expressed efficiently in the P. pastoris expression system. This report may supply a new economic and convenient way for the application of LZ-8 protein.
Eight aerobic bacterial strains were isolated from pulp paper mill waste and screened for tolerance of kraft lignin (KL) using the nutrient enrichment technique in mineral salt media (MSM) agar plate (15 g/L) amended with different concentrations of KL (100, 200, 300, 400, 500, 600 ppm) along with 1% glucose and 0.5% peptone (w/v) as additional carbon and nitrogen sources. The strains ITRC S6 and ITRC S8 were found to have the most potential for tolerance of the highest concentration of KL. These organisms were characterized by biochemical tests and further 16S rRNA gene (rDNA) sequencing, which showed 96.5% and 95% sequence similarity of ITRC S6 and ITRC S8 and confirmed them as Paenibacillus sp. and Bacillus sp., respectively. KL decolorization was routinely monitored with a spectrophotometer and further confirmed by HPLC analysis. Among eight strains, ITRC S6 and ITRC S8 were found to degrade 500 mg/L of KL up to 47.97% and 65.58%, respectively, within 144 h of incubation in the presence of 1% glucose and 0.5% (w/v) peptone as a supplementary source of carbon and nitrogen. In the absence of glucose and peptone, these bacteria were unable to utilize KL. The analysis of lignin degradation products by GC-MS analysis revealed the formation of various acids as lignin monomers which resulted in a decrease in pH and a major change in the chromatographic profile of the bacterial degraded sample as compared to the control clear indications of biochemical modification of KL due to the bacterial ligninolytic system by ITRC S6, namely, acetic acid, propanoic acid, butanoic acid, guaiacol, hexanoic acid, and ITRC S8, namely acetic acid, propanoic acid, ethanedioic acid, furan carboxylic acid, 2-propanoic acid, butanoic acid, 3-acetoxybutyric acid, propanedioic acid, acetoguiacone, 1,2,3-thiadiazole, 5-carboxaldixime, 4-hydroxy-3,5-dimethoxyphenol, and dibutyl phthalate, indicating the bacterium characteristic to degrade G and S units of lignin polymer.