Characterization of a new periplasmic single-domain rhodanese encoded by a sulfur-regulated gene in a hyperthermophilic bacterium Aquifex aeolicus

Rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. Rhodanese domains occur in the three major domains of life. We have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, Aquifex aeolicus, with thiosulfate:cyanide transferase activity, Aq-1599. The oligomeric organization of the enzyme is stabilized by a disulfide bridge. To date this is the first characterization from a hyperthermophilic bacterium of a periplasmic sulfurtransferase with a disulfide bridge. The aq-1599 gene belongs to an operon that also contains a gene for a prepilin peptidase and that is up-regulated when sulfur is used as electron acceptor. Finally, we have observed a sulfur-dependent bacterial adherence linked to an absence of flagellin suggesting a possible role for sulfur detection by A. aeolicus.     

Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH₄⁺ and low concentrations of Co²⁺. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent K_m was calculated to be 4.4 ± 0.07 mM, while V_max was found to be 100 ± 0.02 μM min⁻¹ mg protein⁻¹. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.     

A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus

6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 A, 1.85 A, 2.05 A and 2.2 A, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of single-site mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.     

A transposon-like sequence with short terminal inverted repeats in the nuclear genome of Chlamydomonas reinhardtii

A 1.2 kb DNA sequence, flanked by a potential seven base target-site duplication, was found inserted into a TOC1 transposable element from Chlamydomonas reinhardtii. The insertion sequence, named TOC2, is a member of a family of repeated DNA sequences that is present in all the C. reinhardtii strains tested. It resembles class II transposable elements: it possesses short 14 bp imperfect terminal repeats that begin AGGAGGGT, and sub-terminal direct repeats located within 250 bp of the termini. No large open reading frames were found. The terminal bases and length of target-site duplication are important in classifying transposable elements. On this basis TOC2 does not fall readily into existing families of class II transposable elements found in plants.     

托光柄菇属一中国新记录种

[背景] 托光柄菇属（Volvopluteus）隶属于光柄菇科（Pluteaceae）,目前世界上仅有4个种。[目的] 调查我国东北地区大型真菌资源。[方法] 采集大型真菌标本,对其形态进行详细的观察和描述,提取DNA,测定rDNA ITS序列,基于最大似然法和贝叶斯法构建系统发育树。[结果] 2019–2020年在吉林省延边朝鲜族自治州敦化市采集的标本中,有6份标本为密执安托光柄菇V.michiganensis,该种真菌此前未在我国发现。在系统发育分析中,采自我国的密执安托光柄菇V.michiganensis与该种的模式标本聚为一个分支。[结论] 密执安托光柄菇V.michiganensis为中国新记录种。     

Phylogenetic affiliation of Aeromonas culicicola MTCC 3249(T) based on gyrB gene sequence and PCR-amplicon sequence analysis of cytolytic enterotoxin gene

We determined the gyrB gene sequences of all 17 hybridizations groups of Aeromonas. Phylogenetic trees showing the evolutionary relatedness of gyrB and 16S rRNA genes in the type strains of Aeromonas were compared. Using this approach, we determined the phylogenetic position of Aeromonas culicicola MTCC 3249(T), isolated from midgut of Culex quinquefasciatus. In the gyrB based-analysis A. culicicola MTCC 3249(T) grouped with A. veronii whereas, it grouped with A. jandaei in the 16S rRNA based tree. The number of nucleotide differences in 16S rRNA sequences was less than found with the gyrB sequence data. Most of the observed nucleotide differences in the gyrB gene were synonymous. The Cophenetic Correlation Coefficient (CCC) for gyrB sequences was 0.87 indicating this gene to be a better molecular chronometer compared to 16S rRNA for delineation of Aeromonas species. This strain was found to be positive for the cytolytic enterotoxin gene. PCR-Amplicon Sequence Analysis (PCR-ASA) of this gene showed that the isolate is affiliated to type I and is potentially pathogenic. These PCR-ASA results agreed in part with the gyrB sequence results.     

Morphological discordance of the new trypanosomatid species phylogenetically associated with the genus crithidia

Three new species of monoxenous parasites from the Neotropical Heteroptera are described on the basis of the ultrastructure of cells in culture, as well as gene sequences of Spliced Leader (SL) RNA, glyceraldehyde phosphate dehydrogenase (GAPDH) and small subunit (SSU) rRNA. The results have highlighted a striking discrepancy between the morphological (dis)similarities and the phylogenetic affinities among the insect trypanosomatids. Although each of the new species is characterized by a distinct set of morphological characters, based on the predominant promastigotes observed in culture, each of them has been provisionally assigned to the genus Leptomonas pending the future revision of this genus. Yet, instead of the phylogenetic affinity with the other members of this polyphyletic genus, the new species are most closely related to Crithidia species. Thus, the extremely long promastigotes of Leptomonas acus sp. n. and the unique morphological features found in Leptomonas bifurcata sp. n. sharply contrast with their respective relatives C. fasciculata and C. deanei both of which are typical choanomastigotes. The results clearly show that the current classification at the genus level is misleading and needs to be revised. The phylogenetic clades potentially representing the candidate new genera of monoxenous trypanosomatids have started to emerge from the presented analyses.     

Phylogenetic and genomic analysis of the complete mitochondrial DNA sequence of the spotted asparagus beetle Crioceris duodecimpunctata

We report the complete mitochondrial DNA sequence of the spotted asparagus beetle, Crioceris duodecimpunctata. The genome complement, gene order, and nucleotide composition of this beetle's mitochondrial genome were found to be typical of those reported for other insects. Unusual features of this genome include the substitution of UCU for GCU as the anticodon for tRNA(Ser), an unusual TpsiC loop for the tRNA(Ile) gene, and the identification of a putative ATT start codon for cox1. The utility of complete mitochondrial genome data for phylogenetic inference of the insect orders was tested, and compared to that of cox1 and combined mitochondrial ribosomal DNA sequences. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the orders Diptera, Lepidoptera, and Coleoptera were consistently recovered. Monophyly of the Holometabola was also observed in some (though not all) analyses. The accumulation of complete mitochondrial sequences from a broader array of insect orders holds the promise of clarifying the early diversification of insects.     

Karyotypic and chloroplast genomic diversity inMedicago sect.Lupularia (Leguminosae)

Studies were made on the chromosome complements and chloroplast genomes ofMedicago lupulina andM. secundiflora, which comprise sectionLupularia ofMedicago. Both types of analyses indicated more substantial differences between these species than suggested by external morphology.Medicago lupulina has a relatively asymmetrical karyotype in terms of centromeric position and relative length. The karyotype ofM. secundiflora is comparatively more asymmetrical in centromeric position and reduced in absolute size but exhibits greater symmetry in relative length. The restriction endonuclease fragmentation patterns of the chloropiast DNA of these two species (with Bam HI, Eco RI, Bgl II, and Xho I) show little similarity, with only 17% of the fragments matching in size. The lack of interspecific congruence among data of morphology, karyology and cpDNA inLupularia is contrary to consistency exhibited among these data inMedicago subsect.Intertextae.     

Synonymy of the yeast generaWingea andDebaryomyces

Extent of divergence in partial nucleotide sequences from large and small subunit ribosomal RNAs was used to estimate the evolutionary relationship between the generaWingea andDebaryomyces. These data showed the monotypic genusWingea to be congeneric withDebaryomyces, and it is proposed to transferW. robertsii toDebaryomyces.     

The taxonomic relationships within the genus Excoecaria L. (Euphorbiaceae) based on leaf morphology and rDNA sequence data

The tropical Indo-Pacific genus Excoecaria L. (Euphorbiaceae) has several closely related species in Australia whose taxonomic relationships are unclear. The most widely reported species in Australia is the mangrove species Excoecaria agallocha L. (type species), whose taxonomic and geographic limits are difficult to define from its closely related species or sub-species. Two additional taxa have also been described but not clearly differentiated from the type species: Excoecaria dallachyana Baillon and Excoecaria ovalis Endl. This project aimed to determine the taxonomic relationships of the Australian Excoecaria species using both leaf morphological data and DNA sequence data from the internal transcribed spacer (ITS) region of ribosomal genes. The nucleotide differences in the examined ITS1 region show that E. agallocha from eastern Australia and E. ovalis from Western Australia respectively, are genetically uniform within species but differ from each other consistently, thus supporting species status. The leaf morphological data also support this view: single factor analysis of variance consistently separated E. ovalis from E. agallocha on the basis of leaf width, leaf length and length of petiole. In contrast, E. ovalis from the Gulf of Carpentaria differs only slightly from E. ovalis in Western Australia, but no evidence was found to suggest any leaf morphological differentiation within this species. The analysis also suggests that E. dallachyana is not closely related to either mangrove species E. agallocha or E. ovalis, despite superficial morphological similarities.     

Folding and assembly pathways of co-chaperonin proteins 10: Origin of bacterial thermostability

To compare folding/assembly processes of heptameric co-chaperonin proteins 10 (cpn10) from different species and search for the origin of thermostability in hyper-thermostable Aquifex aeolicus cpn10 (Aacpn10), we have studied two bacterial variants-Aacpn10 and Escherichia coli cpn10 (GroES)-and compared the results to data on Homo sapiens cpn10 (hmcpn10). Equilibrium denaturation of GroES by urea, guanidine hydrochloride (GuHCl) and temperature results in coupled heptamer-to-monomer transitions in all cases. This is similar to the behavior of Aacpn10 but differs from hmcpn10 denaturation in urea. Time-resolved experiments reveal that GroES unfolds before heptamer dissociation, whereas refolding/reassembly begins with folding of individual monomers; these assemble in a slower step. The sequential folding/assembly mechanism for GroES is rather similar to that observed for Aacpn10 but contradicts the parallel paths of hmcpn10. We reveal that Aacpn10's stability profile is shifted upwards, broadened, and also moved horizontally to higher temperatures, as compared to that of GroES.     

General Stress Protein CTC from Bacillus subtilis Specifically Binds to Ribosomal 5S RNA

Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.     

Intersubunit Disulfide Interactions Play a Critical Role in Maintaining the Thermostability of Glucose-6-phosphate Dehydrogenase from the Hyperthermophilic Bacterium Aquifex aeolicus

Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t_1/2=24 hr at 90°C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine 184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines. The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90°C. The same decrease in half-life was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability of the enzyme.     

The phylogeny of the genus Ischioscia Verhoeff, 1928, with redescriptions of three species (Crustacea: Isopoda: Oniscidea)

The genus Ischioscia Verhoeff, 1928 is reviewed. 26 species are considered valid. A key for their identification is given, as well as a map showing the geographic distribution. The known range of the genus covers a large area from the central Amazon region to the mountains of Guatemala. The species of Ischioscia have a typical “philosciid” habitus (“runner” type); they can be distinguished from other Neotropical species with similar habitus by the following apomorphies: (1) male pereiopod 1 carpus enlarged to a plate-like extension, (2) scale field on male pereiopod 1 covering entire frontal side of the carpus, (3) male pereiopod 7 ischium with a ventral scale field, (4) dactylus in both sexes with a long inner claw. The groundpattern of Ischioscia is reconstructed, and an analysis of the phylogenetic relations within the genus is made on the basis of morphological data. The species are very similar to each other, most differences are found in the male structures of sexually dimorphic features. Ischioscia sturmi (Vandel, 1972), I. amazonica Lemos de Castro, 1955 and I. bolivari Vandel, 1968 are redescribed in detail.     

Reclassification of Bacteroides putredinis (Weinberg et al., 1937) in a new genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp. nov., from human sources

During studies on the bacteriology of appendicitis in children, we often isolated from inflamed and non-inflamed tissue samples, an unusual bile-resistant pigment-producing strictly anaerobic gram-negative rod. Phenotypically this organism resembles members of Bacteroides fragilis group of species, as it is resistant to bile and exhibits a special-potency-disk pattern (resistance to vancomycin, kanamycin and colistin) typical for the B. fragilis group. However, the production of brown pigment on media containing haemolysed blood and a cellular fatty acid composition dominated by iso-C15:0, suggests that the organism most closely resembles species of the genus Porphyromonas. However, the unidentified organism differs from porphyromonads by being bile-resistant and by not producing butyrate as a metabolic end-product. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism represents a distinct sub-line, associated with but distinct from, the miss-classified species Bacteroides putredinis. The clustering of the unidentified bacterium with Bacteroides putredinis was statistically significant, but they displayed > 4% sequence divergence with each other. Chromosomal DNA-DNA pairing studies further confirmed the separateness of the unidentified bacterium and Bacteroides putredinis. Based on phenotypic and phylogenetic considerations, it is proposed that Bacteroides putredinis and the unidentified bacterium from human sources be classified in a new genus Alistipes, as Alistipes putredinis comb. nov. and Alistipes finegoldii sp. nov., respectively. The type strain of Alistipes finegoldii is CCUG 46020(T) (= AHN243(T)).     

A reconsideration of the genusRabdosiella (Lamiaceae,Nepetoideae, Ocimeae)

The two generaPlectranthus andIsodon are compared and found to be very dissimilar.Isodon ist considered to be misplaced inOcimeae subtribePlectranthinae and apparently is more closely related to subtribeHyptidinae. The disjunct genusRabdosiella is compared to these two genera and regarded to be polyphyletic. The AfricanR. calycina (Benth.)Codd is returned toPlectranthus and calledP. calycinusBenth., while the AsianR. ternifolia (D. Don)Codd is placed inIsodon sect.Pyramidium and calledI. ternifolius (D. Don)Kudo.     

A relationship between free amino acid and nickel contents in leaves and seeds ofAlyssum bertolonii

Summary Data are given on the aqueous acetone—soluble (free) amino acid composition and total nickel contents of leaves of wild and cultivatedAlyssum bertolonii and of seeds produced by the former. Leaves of the wild plants show a direct relationship between their total free amino acid N and Ni contents. These are highest in February, show a decrease in June and increase again by October. It is inferred that variations in the Ni uptake and free amino acid contents are functions of seasonal factors. Leaves of cultivated plants showed the lowest Ni content but the highest free amino acid content illustrating the influence of environmental factors.     

Morphological and ITS1, 5.8S,and Partial ITS2 Ribosomal DNA Sequence Distinctions Between Two Species <Emphasis Type="Italic">Playtygyra</Emphasis> (Cnidaria: Scleractinia) from Hong Kong

Two sympatric species of Platygyra have been identified from Hong Kong waters: i.e., P. sinensis and P. pini. The former has been further subdivided into 4 morphotypes based on colony growth form as follows: classic, encrusting, hillocky, and long-valley. Taxonomic confusion raised by overlapping morphological variations and frequent sympatric occurrences, however, has posed problems in relation to Platygyra ecology and population dynamics. This study attempted to differentiate Platygyra pini and morphotypes of P. sinensis by both morphological and ITS1, 5.8S, and partial ITS2 ribosomal DNA sequence analysis. Morphological data based on 9 skeletal characters were subjected to multivariate analysis. No clear groupings were obtained using a multidimensional scaling plot. Most parsimony analysis was conducted using either the rDNA data set including ITS1, 5.8S, and partial ITS2 or the ITS1 region only. Maximum parsimony (MP) and neighbor-joining (NJ) trees obtained from both data sets, clustered samples of P. sinensis and P. pini into 2 clades. The interspecific Kimura 2-parameter sequence divergence value (k2) obtained by the former rDNA data set was 14.275 ± 0.507%, which is greater than the intraspecific values (1.239 ± 1.147% for P. sinensis and 0.469 ± 0.364% for P. pini), indicating that this marker of ITS1, 5.8S, and ITS2 contains substantially high levels of inherent diversity and is useful in resolving the problematic taxonomy of Platygyra.