Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.     

Triquetrella mxinwana,a new moss species from South Africa,with a phylogenetic and biogeographic hypothesis for the genus

Abstract

We describe Triquetrella mxinwana, a new species presently known only from the winter rainfall area of South Africa. Within this region it is common in karroo and renosterveld, but it also occurs in fynbos. It differs from other Triquetrella species in the several low, bifid, papillae on each laminal cell, but is similar to the Australian Leptodontium paradoxum, differing in size and anatomical details. Although sporophytes of L. paradoxum are unknown, those of T. mxinwana have the peristome of Triquetrella rather than Leptodontium. Phylogenetic analysis of data from three chloroplast (rps4, trnL-F and psbA-trnH) and one nuclear (ITS1) loci confirms that T. mxinwana and L. paradoxum are sister taxa and together are sister to the rest of Triquetrella. This placement also better reflects gametophyte morphology, which, though intermediate between Triquetrella and Leptodontium, better fits the former. Divergence levels among species of Triquetrella are very low and molecular clock approaches indicate that all divergences are of Pliocene–Pleistocene age. The estimated time of the split between T. mxinwana and its nearest relative is concordant with independent paleoclimatic estimates of the time of onset of winter rainfall conditions in the Cape. The disjunctive distribution of the genus in areas of Mediterranean climate is attributed to dispersal rather than ancient vicariance.     

NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda)

Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit I sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (5.9–30.8%) was usually greater than in cytochrome c oxidase subunit I (2.5–18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.     

Oligonucleotide fingerprinting of Hela cell 5 S RNA labeled in vitro with 125I

HeLa cell 5 S ribosomal RNA, labeled in vitro at 40°, 60°, or 70°C with ¹²⁵I, has been digested with RNase T1 and fingerprinted by conventional methods. Regardless of the temperature of iodination, ¹²⁵I was bound (exclusively) to virtually all CMP residues. Characterization of labeled oligonucleotides yielded sequence information consistent with that derived from in vivo labeled RNA.     

The Constituents of the Essential Oil from Litsea japonica (Thunb.) Juss., FRUIT

Tetragenococcus halophila D10 decarboxylates aspartate to alanine, but T. halophila D10 derivatives generated by a curing treatment could not (Asd^- derivatives). We observed by electrophoresis three plasmid bands in T. halophila D10; all Asd^- derivatives lost the largest of these bands. This plasmid, pD1, has two SalI sites. We cloned and sequenced the 10 kb SalI fragment. The DNA sequence suggests that this fragment contains the aspartate decarboxylating trait.     

Neospora caninum:Tachyzoites Express a Potent Type-I Nucleoside Triphosphate Hydrolase,but Lack Nucleoside Diphosphate Hydrolase Activity

Asai, T., Howe, D. K., Nakajima, K., Nozaki, T., Takeuchi, T., and Sibley, L. D.Neospora caninum: Tachyzoites Express Type-I Nucleoside Triphosphate Hydrolase1. But Lack Nucleoside Diphosphate Hydrolase Activity.Experimental Parasitology90,277–285. We have identified type I nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) activity, previously thought to be restricted to the virulent strains ofToxoplasma gondii, in the cell extracts ofNeospora caninumtachyzoites. Sequence analysis of a complete cDNA from Nc-1 strain indicated thatN. caninumNTPases shared approximately 69% identity to the NTPases ofT. gondiiand are most similar to the NTPase-I isozyme. Southern blot analysis of genomic DNA and sequence analysis of two independentNTPclones from the Nc-1 strain revealed the presence of multiple genes, at least two of which are transcribed. Substrate specificity andK_mvalues for MgATP²⁻and MgADP⁻hydrolysis for recombinant or partially purified native NcNTPase were the same as those for the type I isozyme (NTPase-I). Significantly, no type II enzyme (NTPase-II) activity for NDP hydrolysis was detected in cell extracts ofN. caninum, although it is universally present in allT. gondiistrains that have been tested. This intriguing difference between these two closely related apicomplexan parasites may provide insight into the function of the NTPases during intracellular parasitism.     

Isolation,Functional Characterization,and Expression Pattern of a Vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">TrNHX1</Emphasis> from <Emphasis Type="Italic">Trifolium repens</Emphasis> L.

Plant vacuolar Na⁺/H⁺ antiporter plays an important role in salt tolerance. A vacuolar Na⁺/H⁺ antiporter gene TrNHX1 was cloned from Trifolium repens L., a forage legume, by RT-PCR and RACE methods using degenerate oligonucleotide primers. The TrNHX1 sequence contains 2,394 nucleotides and an open-reading frame of 1,626 nucleotides that encodes a protein of 541 amino acids with a deduced molecular mass of 59.5 kDa. The deduced amino acid sequence of TrNHX1 is 78% identical to that of a vacuolar Na⁺/H⁺ antiporter of Arabidopsis thaliana, AtNHX1, and contains the consensus amiloride-binding domain. TrNHX1 could partially complement the NaCl-sensitive phenotypes of yeast mutants Δnhx1 and Δena1-4Δnhx1, and a similar complementation was also observed in the presence of LiCl and KCl. In addition, it was found that TrNHX1 suppressed the hygromycin-sensitive phenotype of yeast mutant Δena1-4Δnhx1. The expression of TrNHX1 in T. repens increased in the presence of 150 mM NaCl, and this result accords with that of Na⁺ contents determination under the same treatment. These results suggest that TrNHX1 functions as a vacuolar Na⁺/H⁺ antiporter and plays an important role in salt tolerance and ion homeostasis in T. repens.     

A fine structure map of spontaneous and induced mutations in the lambda repressor gene, including insertions of IS elements

Summary Mutations at over 70 sites in the cI gene have been mapped by 4-factor crosses and assigned precise or approximate positions in the DNA sequence. 16 of 25 spontaneous mutations were insertions of IS1, IS3 or IS5 into AT-rich regions of cI. The 5-methylcytosine in the sequence Cm⁵CAGG is a hot spot for spontaneous cI amber mutations. Recombination frequencies between mutations were proportional to distance with the exception of amber mutations at 4 sites, including the host spot for spontaneous mutations. Mutations with a given phenotype are clustered on the genetic map. No missense mutations affecting repressor activity were found in the central one-third of cI, but 5 of 6 ind ^- mutations were located in this region. The aminoterminal third of the gene contains the sites of most trans-dominant cI^- mutations, and of all ts mutations that result in repressors that are reversibly inactivated at high temperatures.     

Systematic status and phylogenetic relationships of the enigmatic Tanacetum paradoxum Bornm. (Asteraceae, Anthemideae): evidences from nrDNA ITS, micromorphological, and cytological data

In order to evaluate the phylogenetic relationships and taxonomic classification of the Iranian endemic Tanacetum paradoxum Bornm. (Asteraceae, Anthemideae), a molecular analysis based on nrDNA ITS sequences of 16 species of Tanacetum together with representatives of 27 genera of subtribes Artemisiinae and Handeliinae as well as a micromorphological study of achenes and pollen grains was performed. The chromosome number of 2n?=?2x?=?16 and the genome size of T. paradoxum (2C?=?8.81?±?0.19?pg) are reported here for the first time. Due to the well-supported close phylogenetic relationship of T.?paradoxum to the species of the genus Artemisia (Bayesian inference, posterior probability PP?=?1), which is supported by cytological (x?=?8) and micromorphological features concerning the achene shape and pollen type, the combination Artemisia paradoxa (Bornm.) Sonboli is proposed.     

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

Summary The nucleotide sequence of a 1105 by Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA^Gln (CUG) genes, differing by 1 by in the aminoacyl stem, and three identical tRNA^Glu (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glul-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3 terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3 terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.     

Seasonal dynamics of zooplankton in a shallow eutrophic,man-made hyposaline lake in Delhi (India): role of environmental factors

Old Fort Lake, a small (1.6 ha), shallow, and recreational water body in Delhi (India) was studied through monthly surveys in two consecutive years (January, 2000–December, 2001). Precipitation is the major source of water for this closed basin lake. In addition, ground water is used for replenishing the lake regularly. This alkaline, hyposaline hard water lake contains very high ionic concentration, especially of nitrates. Based on overall ionic composition, this lake can be categorized as chloride–sulfate alkaline waters with the anion sequence dominated by SO₄ ²⁻ > Cl⁻ > HCO₃ ⁻, and the cations by Mg⁺⁺ > Ca⁺⁺. The overall seasonal variability in physicochemical profile was largely regulated by the annual cycle of evaporation and precipitation, whereas the ground water largely influences its water quality. The lake exhibited phytoplankton-dominated turbid state due to dominance of the blue green alga, Microcystis aeruginosa. The persistent cyanobacterial blooms and the elevated nutrient levels are indicative of the cultural eutrophication of the lake. This study focuses on the relative importance of eutrophic vis-à-vis hyposaline conditions in determining the structure and seasonal dynamics of zooplankton species assemblages. A total of 52 zooplankton species were recorded and rotifers dominated the community structure qualitatively as well as quantitatively. The genus Brachionus comprised a significant component of zooplankton community with B. plicatilis as the most dominant species. The other common taxa were B. quadridentatus, B. angularis, Lecane grandis, L. thalera, L. punctata, Mesocyclops sp., and Alona rectangula. Multivariate data analysis techniques, Canonical Correspondence Analysis (CCA) along with Monte Carlo Permutation Tests were used to determine the minimum number of environmental factors that could explain statistically significant (P < 0.05) proportions of variation in the species data. The significant variables selected by CCA were NH₃–N followed by percent saturation of DO, COD, SS, BOD, NO₂–N, rainfall, silicates, and PO₄–P. The results indicate that the seasonal succession patterns of the zooplankton species were largely controlled by physicochemical factors related directly or indirectly to the process of eutrophication, whereas hyposaline conditions in the lake determined the characteristic species composition. Guest Editors: J. John & B. Timms Salt Lake Research: Biodiversity and Conservation—Selected papers from the 9th Conference of the International Society for Salt Lake Research     

Phylogeny of the genera Trichophyton using mitochondrial DNA analysis

Diversity of mitochondrial DNA (mtDNA) was investigated in 92 Trichophyton rubrum strains, 2 T. mentagrophytes var. mentagrophytes, 2 T. m. vor. interdigitale, 2 T. m. var. goetzii, 1 T. m. var. erinacei, 2 T. quinckeanum, 2 T. schoenleinii, 1 T. tonsurans, 2 T. verrucosum var. album, 2 T. v. var. discoides, 1 T. violaceum var. violaceum, 1 Arthroderma benhamiae, and 1 A. vanbreuseghemii using endonucleases, Hae III, Msp I, Hind III, Xba I, and Bgl II. Trichophyton species were divided into 7 groups, and a phylogenetic tree was produced based on sequence divergence within mtDNA. The following results were obtained: (1) T. rubrum was divided into 2 groups Type I and Type II, and was suggested to be a complex. (2) A. benhamiae was closely related to T. m. var. erinacei. (3) T. rubrum Type II, T. tonsurans, and A. vanbreuseghemii showed identical restriction profiles, and were suggested to be closely related to each other or identical. (4) T. quinckeanum and T. schoenlenii showed identical restriction profiles, which differed slightly from those of A. vanbreuseghemii. (5) mtDNA analysis was useful in identifying pleomorphic strains.     

Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse

 A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2 ^bc ). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2 ^k , H2 ^b , and H2 ^bc , and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6-Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species. Received: 27 April 1998 / Revised: 4 June 1998     

DNA sequence of a class I pseudogene from theTla region of the murine MHC: Recombination at a B2 Alu repetitive sequence

Summary We have determined the DNA sequence of a BALB/cTla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1^c gene from theTla ^c genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1^c gene contains sequences that induced expression of a serologically reactiveTla gene in the genome of the recipient L cell. The T1^c gene is structurally related to the previously sequenced T13^c gene that encodes a serologically defined TL antigen. The 3 half of the T1^c gene including exons 4, 5, 6, and the 3 untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13^c gene; however, the 5 half of the T1^c gene has little homology with the T13^c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13^c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.     

Jeotgalibacillus aurantiacus sp. nov., a novel orange-pigmented species with a carotenoid biosynthetic gene cluster,isolated from wetland soil

A Gram-stain-positive, orange-pigmented, rod-shaped and flagellated bacterial strain T12^T was isolated from wetland soil in Kunyu Mountain Wetland in Yantai, China. The strain was able to grow at 15–40 °C (optimum 37 °C), at 0.0–9.0% NaCl (optimum 2%, w/v) and at pH 5.5–9.0 (optimum 8.5). A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain T12^T is a member of the family Planococcaceae, sharing 97.6% and 97.1% sequence similarity with the type strains of Jeotgalibacillus salarius and Jeotgalibacillus marinus, respectively. Genome-based analyses revealed a genome size of 3,506,682 bp and a DNA G?+?C content of 43.7%. Besides, the genome sequence led to 55.0–74.6% average amino acid identity values and 67.8–74.7% average nucleotide identity values between strain T12^T and the current closest relatives. Digital DNA-DNA hybridization of strain T12^T with the type strains of Jeotgalibacillus proteolyticus and J. marinus demonstrated 19.0% and 20.3% relatedness, respectively. The chemotaxonomic analysis showed that the sole quinone was MK-7. The predominant cellular fatty acids were iso-C_15:0, anteiso-C_15:0, C_16:1ω7c alcohol and iso-C_14:0. The polar lipids consisted of an unidentified aminolipid, phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. Based on the polyphasic characterization, strain T12^T is considered to represent a novel species, for which the name Jeotgalibacillus aurantiacus sp. nov. is proposed. The type strain is T12^T (=?KCTC 43296 ^T?=?MCCC 1K07171^T).

     

Nucleotide sequence analysis of IS427 and its target sites inAgrobacterium tumefaciens T37

We have determined the nucleotide sequence of IS427, an insertion sequence fromAgrobacterium tumefaciens T37. IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication. It is present at three sites in the pTiT37 plasmid and is absent from the chromosome ofA. tumefaciens T37. Each of the IS427 elements sequenced was near a site with sequence homology to integration host factor (IHF)-binding sites which suggested that IHF may be involved in IS427 transposition.