Exiguobacterium algae sp. nov. and Exiguobacterium qingdaonense sp. nov., two novel moderately halotolerant bacteria isolated from the coastal algae

Antonie van Leeuwenhoek - Two Gram-stain-positive, facultatively anaerobic, rod-shaped bacterial strains, S126T and S82T, were isolated from coastal algae of China. Strains S126T and S82T are...     

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32% approximately 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, AfDnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana.     

Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemia franciscana

SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and flies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemia franciscana. The amino acid sequence identity between the Artemia and the fly SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemia franciscana provides intriguing features of the divergent protein, SPARC.     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Halopenitus salinus sp. nov., isolated from the brine of salted brown alga Laminaria

A halophilic archaeal strain, SKJ47^T, was isolated from a commercial preparation of the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells of the strain were observed to be short rods, stain Gram-negative, and to form red-pigmented colonies on solid media. Strain SKJ47^T was found to be able to grow at 20–50 °C (optimum 37 °C), at 0.9–4.8 M NaCl (optimum 2.6–3.1 M), at pH 6.0–9.5 (optimum pH 7.0). The cells lysed in distilled water and the minimal NaCl concentration to prevent cell-lysis was found to be 5 % (w/v). The major polar lipids of the strain were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and two glycolipids chromatographically identical to those of Halopenitus persicus IBRC 10041^T. The 16S rRNA gene and rpoB′ gene of strain SKJ47^T were found to be phylogenetically related to the corresponding genes of Halopenitus malekzadehii IBRC-M 10418^T (96.3 and 91.9 % nucleotide identity, respectively) and Hpt. persicus IBRC 10041^T (96.2 and 93.8 %). The DNA G+C content of strain SKJ47^T was determined to be 65.0 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain SKJ47^T (=CGMCC 1.12229^T = JCM 18641^T) represents a new species of the genus Halopenitus, for which the name Halopenitus salinus sp. nov. is proposed.     

Mycoplasma simbae sp. nov., Mycoplasma leopharyngis sp. nov., and Mycoplasma leocaptivus sp. nov., isolated from lions.

Mycoplasmas isolated from the throats of lions were shown to belong to three serotypes, all of which were serologically distinct from the previously recognized Mycoplasma and Acholeplasma spp. Eight mycoplasma colonies were cloned, including one from a leopard (strain LP), and were examined in detail for morphology, growth, and biochemical characteristics. The strains had the following properties: guanine-plus-cytosine contents of 37 mol% (strain LXT [T = type strain]), 28 mol% (strain LL2T), and 27 mol% (strain 3L2T) and a requirement for sterol. Strain 3L2T metabolized glucose, which was not metabolized by strains LXT and LL2T. Arginine and urea were not hydrolyzed. Strain LX (= NCTC 11724) is the type strain of a new species, Mycoplasma simbae; strain LL2 (= NCTC 11725) is the type strain of a second new species, Mycoplasma leopharyngis; and strain 3L2 (= NCTC 11726) is the type strain of a third new species, Mycoplasma leocaptivus.     

Exiguobacterium himgiriensis sp. nov. a novel member of the genus Exiguobacterium, isolated from the Indian Himalayas

The taxonomic position of an orange coloured bacterium, strain K22–26^T isolated from a soil sample was studied using a polyphasic approach. The organism had phenotypic and chemotaxonomic properties consistent with its allocation into the genus Exiguobacterium. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain K22–26^T belongs to the genus Exiguobacterium and was related to Exiguobacterium aurantiacum DSM 6208^T (99.0 %) Exiguobacterium mexicanum DSM 16483^T (98.6 %), Exiguobacterium aquaticum (98.6 %), Exiguobacterium aestuarii DSM 16306^T (98.1 %), Exiguobacterium profundum DSM 17289^T (98.1 %) and Exiguobacterium marinum DSM 16483^T (97.9 %), whereas sequence similarity values with respect to other Exiguobacterium species with validly published names were between 92.5–94.0 %. The major polar lipids detected were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major menaquinone was determined to be MK-7 (83 %) whereas MK-8 (11 %) and MK-6 (6 %) occur in smaller amounts. The peptidoglycan of the strain was found to contain l-lysine as the diagnostic diamino acid. The major fatty acids detected were iso C_13:0 (11.2 %), anteiso C_13:0 (15.4 %), iso C_15:0 (13.2 %) and iso C_17:0 (16.1 %). However, analysis of the DNA–DNA relatedness confirmed that strain K22–26^T belongs to a novel species. The G + C content of the strain K22–26^T was determined to be 50.1 mol %. The novel strain was distinguished from closely related type species of the genus Exiguobacterium using DNA–DNA relatedness and phenotypic data. Based on these differences, the strain K22–26^T should be classified as a novel species of the genus Exiguobacterium, for which the name Exiguobacterium himgiriensis sp. nov. strain K22–26^T (= MTCC 7628^T = JCM 14260^T) is proposed.     

Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.     

Iodidimonas gelatinilytica sp. nov., aerobic iodide-oxidizing bacteria isolated from brine water and surface seawater

Antonie van Leeuwenhoek - Chemo-organotrophic iodide (I?)-oxidizing bacterial strains Hi-2T and Mie-1 were isolated from iodide-rich natural gas brine water in Chiba and surface seawater in...     

Chryseobacterium zeae sp. nov., Chryseobacterium arachidis sp. nov., and Chryseobacterium geocarposphaerae sp. nov. isolated from the rhizosphere environment

Four yellow pigmented strains (91A-561^T, 91A-576, 91A-593^T, and JM-1085^T) isolated from plant materials, showed 97.2–98.7 % 16S rRNA gene sequence similarities among each other and were studied in a polyphasic approach for their taxonomic allocation. Cells of all four isolates were rod-shaped and stained Gram-negative. Comparative 16S rRNA gene sequence analysis showed that the four bacteria had highest sequence similarities to Chryseobacterium formosense (97.2–98.7 %), Chryseobacterium gwangjuense (97.1–97.8 %), and Chryseobacterium defluvii (94.6–98.0 %). Sequence similarities to all other Chryseobacterium species were below 97.5 %. Fatty acid analysis of the four strains showed Chryseobacterium typical profiles consisting of major fatty acids C_15:0 iso, C_15:0 iso 2-OH/C_16:1 ω7c, C_17:1 iso ω9c, and C_17:0 iso 3-OH, but showed also slight differences. DNA–DNA hybridizations with type strains of C. gwangjuense, C. formosense, and C. defluvii resulted in values below 70 %. Isolates 91A-561^T and 91A-576 showed DNA–DNA hybridization values >80 % indicating that they belonged to the same species; but nucleic acid fingerprinting showed that the two isolates represent two different strains. DNA–DNA hybridization results and the differentiating biochemical and chemotaxonomic properties showed, that both strains 91A-561^T and 91A-576 represent a novel species, for which the name Chryseobacterium geocarposphaerae sp. nov. (type strain 91A-561^T=LMG 27811^T=CCM 8488^T) is proposed. Strains 91A-593^T and JM-1085^T represent two additional new species for which we propose the names Chyrseobacterium zeae sp. nov. (type strain JM-1085^T=LMG 27809^T, =CCM 8491^T) and Chryseobacterium arachidis sp. nov. (type strain 91A-593^T=LMG 27813^T, =CCM 8489^T), respectively.     

Metschnikowia noctiluminum sp. nov., Metschnikowia corniflorae sp. nov., and Candida chrysomelidarum sp. nov., isolated from green lacewings and beetles

Fourteen yeast isolates belonging to the Metschnikowia clade were isolated from the digestive tracts of lacewings (Neuroptera: Chrysopidae), soldier beetles and leaf beetles (Coleoptera: Cantharidae and Chrysomelidae), and a caddisfly (Trichoptera: Hydropsychidae). The insect hosts were associated with sugary substances of plants, a typical habitat for yeasts in this clade. Based on DNA sequence comparisons and phenetic characters, the yeasts were identified as Candida picachoensis, Candida pimensis, and four undescribed taxa. Among the undescribed taxa, three yeasts were distinguished from one another and from other described taxa by nucleotide differences in the ribosomal DNA repeat, which were sufficient to consider them as new species. Two of the novel yeast species are described as Metschnikowia noctiluminum (NRRL Y-27753^T) and M. corniflorae spp. nov. (NRRL Y-27750^T) based in part on production of needle-shaped ascospores, which are found in most Metschnikowia species. Sexual reproduction was not observed in the third new yeast, Candida chrysomelidarum sp. nov. (NRRL Y-27749^T). A fourth isolate, NRRL Y-27752, was not significantly distinct from Metschnikowia viticola and Candida kofuensis to be described as a new species. Phylogenetic analysis of the D1/D2 loop sequences placed M. noctiluminum within the M. viticola clade, while C. chrysomelidarum was a sister taxon of Candida rancensis. Metschnikowia corniflorae was phylogenetically distinct from other new species and fell outside of the large-spored Metschnikowia group.     

Allobaculum stercoricanis gen. nov., sp. nov., isolated from canine feces

Morphological, biochemical, and molecular genetic studies were performed on an unknown anaerobic, catalase-negative, non-spore-forming, rod-shaped bacterium isolated from dog feces. The unknown bacterium was tentatively identified as a Eubacterium species, based on cellular morphological and biochemical tests. 16S rRNA gene sequencing studies, however, revealed that it was phylogenetically distant from Eubacterium limosum, the type species of the genus Eubacterium. Phylogenetically, the unknown species forms a hitherto unknown sub-line proximal to the base of a cluster of organisms (designated rRNA cluster XVI), which includes Clostridium innocuum, Streptococcus pleomorphus, and some Eubacterium species. Based on both phenotypic and phylogenetic criteria, it is proposed that the unknown bacterium be classified as a new genus and species, Allobaculum stercoricanis. Using a specific rRNA-targeted probe designed to identify Allobaculum stercoricanis, in situ hybridisation showed this novel species represents a significant organism in canine feces comprising between 0.1% and 3.7% of total cells stained with DAPI (21 dog fecal samples). The type strain of Allobaculum stercoricanis is DSM 13633(T)=CCUG 45212(T).     

Chitinimonas taiwanensis gen. nov., sp. nov., a novel chitinolytic bacterium isolated from a freshwater pond for shrimp culture

A bacterial strain, designated cfT was isolated from surface water of a freshwater pond for shrimp (Macrobrachium rosenbergii) culture at Ping-Tung (Southern Taiwan). Cells of this organism were Gram-negative, slightly curved rods which were motile by means of a single polar flagellum. Strain cfT utilized chitin as the exclusive carbon, nitrogen, and energy source for growth, both under aerobic and anaerobic conditions. Optimum conditions for growth were between 25 and 37 degrees C, 0 and 1% NaCl and pH 6 to 8. Strain cfT secreted two chitinolytic enzymes with approximate molecular weight 52 and 64 kDa, which hydrolyzed chitin to produce chitotriose as major product. Sequence comparison of an almost complete 16S rDNA gene showed less than 92% sequence similarity with known bacterial species. Phylogenetic analysis based on the neighbour-joining and other methods indicated that the organism formed a distinct lineage within the beta-subclass of Proteobacteria. The predominant cellular fatty acids of strain cfT were hexadecanoic acid (about 29%), octadecenoic acid (about 12%) and summed feature 3 (16:1 omega7c or 15 iso 2-OH or both [about 49%]). Its DNA base ratio was 62.8 mol% G+C. We propose to classify strain cfT (= CCRC 17210T = LMG 22011T) as Chitinimonas taiwanensis gen. nov., sp. nov.