Dietzia natronolimnaios sp. nov., a new member of the genus Dietzia isolated from an East African soda lake

Two novel alkaliphilic aerobic organotrophic bacteria have been isolated from a moderately saline and alkaline East African soda lake. The new isolates grow at pH values between 6 and 10, with a pH optimum for growth of 9.0, and at a salt concentration between 0% and 10% (w/v). Phylogenetic analysis based on 16S rDNA sequence shows that these isolates are very closely related (99.6% similarity) and are members of the monospecific genus Dietzia (98.8% and 98.7% similarity). DNA/DNA hybridization revealed a relatedness of 83% between the two isolates, but only 8% between them and the type strain Dietzia maris. The G + C content as measured by thermal denaturation is 66.1 mol%. Phenotypic comparisons between D. maris and one isolate showed that they share very similar morphological and chemotaxonomic properties, but differ significantly in carbon source utilization profiles and halotolerance in alkaline medium. We propose a second species of this genus which we name Dietzia natronolimnaios (type strain 15LN1 = CBS 107.95). Received: October 14, 1997 / Accepted: February 26, 1998     

Effect on the Medium Salinity on Oil Degradation by Nocardioform Bacteria

Oil degradation by cultures of Rhodococcus erythropolisand Dietzia mariswas found to depend on the NaCl concentration in the medium. Optimal utilization of turbine oil by R. erythropolisand D. mariswas observed at 0.5 and 2 to 5% NaCl concentration, respectively. Mineral oil and a mixture of paraffins (C₁₄–C₁₈) were utilized within a broader range of the medium salinity. As shown by fluorescent microscopy, D. mariscolonies formed on the oil drop surface, whereas R. erythropoliscells penetrated the drops. The strains studied may populate various ecological niches in oil-containing ecosystems. They are promising for the development of microbial preparations for cleaning the environment from oil pollution.     

Bacterial Endosymbionts of Pyrodinium bahamense var. compressum

The study presents evidence in support of the bacterial theory associated with the toxicity of Pyrodinium bahamense var. compressum. Bacterial endosymbionts from Philippine P. bahamense var. compressum strain Pbc MZRVA 042595 were isolated and identified via 16S rDNA sequence analysis. Taxonomic diversity of the identified culturable intracellular microbiota associated with Philippine P. bahamense var. compressum was established to be limited to the Phyla Proteobacteria, Actinobacteria, and Firmicutes. Major endosymbionts identified included Moraxella spp., Erythrobacter spp., and Bacillus spp., whereas Pseudomonas putida, Micrococcus spp., and Dietzia maris were identified as minor isolates. All identified strains except D. maris, P. putida, and Micrococcus spp. were shown to contain either saxitoxin or neo saxitoxin or both at levels ≤73 ng/10⁷ bacterial cells based on high-performance liquid chromatography analysis. Paralytic shellfish poisoning-like physiologic reactions in test animals used in the mouse assay were recorded for the endosymbionts except for P. putida. The study is the first to elucidate the possible contribution of bacterial endosymbionts in the toxicity of P. bahamense var. compressum isolated in the Philippines.     

A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10⁸ to 10⁹ per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

Alkane‐degrading properties of Dietzia sp. H0B,a key player in the Prestige oil spill biodegradation (NW Spain)

Aims: Investigation of the alkane‐degrading properties of Dietzia sp. H0B, one of the isolated Corynebacterineae strains that became dominant after the Prestige oil spill. Methods and Results: Using molecular and chemical analyses, the alkane‐degrading properties of strain Dietzia sp. H0B were analysed. This Grampositive isolate was able to grow on n‐alkanes ranging from C₁₂ to C₃₈ and branched alkanes (pristane and phytane). 8‐Hexadecene was detected as an intermediate of hexadecane degradation by Dietzia H0B, suggesting a novel alkane‐degrading pathway in this strain. Three putative alkane hydroxylase genes (one alkB homologue and two CYP153 gene homologues of cytochrome P450 family) were PCR‐amplified from Dietzia H0B and differed from previously known hydroxylase genes, which might be related to the novel degrading activity observed on Dietzia H0B. The alkane degradation activity and the alkB and CYP153 gene expression were observed constitutively regardless of the presence of the substrate, suggesting additional, novel pathways for alkane degradation. Conclusions: The results from this study suggest novel alkane‐degrading pathways in Dietzia H0B and a genetic background coding for two different putative oil‐degrading enzymes, which is mostly unexplored and worth to be subject of further functional analysis. Significance and Impact of the Study: This study increases the scarce information available about the genetic background of alkane degradation in genus Dietzia and suggests new pathways and novel expression mechanisms of alkane degradation.     

Characterisation of a lipomannan lipoglycan from the mycolic acid containing actinomycete Dietzia maris

Lipoglycans such as the mycobacterial lipoarabinomannans (LAM) are important cell envelope components of actinomycetes. To further our understanding of the diversity of these enigmatic macromolecules the lipoglycan composition of Dietzia maris has been investigated. Phenol-water extraction and hydrophobic interaction chromatography were used to purify a lipoglycan which was unusually small and predominantly lipomannan in nature. The presence of minor levels of arabinose along with components consistent with the presence of a phosphatidylinositol anchor suggest that this lipoglycan is a novel representative of the lipomannan/LAM structural archetype. This was further supported by the observed cross-reaction of the D. maris lipoglycan with an antiserum raised against LAM from Mycobacterium tuberculosis. These findings reveal a previously unsuspected diversity in the lipoglycan composition of the mycolic acid containing actinomycetes and are further discussed in relation to the apparent absence of phosphatidylinositolmannoside glycolipids in D. maris.     

Description of Dietzia lutea sp. nov., isolated from a desert soil in Egypt

An actinobacterial strain YIM 80766^T was isolated from a soil sample collected from the eastern desert of Egypt, and its taxonomic position was investigated by a polyphasic approach. The organism was found to have a range of chemical and morphological properties consistent with its classification in the genus Dietzia. Phylogenetic analysis indicated that the levels of 16S rRNA gene sequence similarity between strain YIM 80766^T and the other type strains of recognized members of the genus Dietzia were 97.0–98.9%. However, DNA–DNA hybridization values and phenotypic characteristics revealed that the strain differed from the currently recognized species of the genus Dietzia. Therefore, strain YIM 80766^T represents a novel species of the genus Dietzia, for which the name Dietzia lutea sp. nov. is proposed. The type strain is YIM 80766^T (=KCTC 19232^T=DSM 45074^T=CCTCC AA 207008^T). The 16S rRNA gene sequence of strain YIM 80766^T has been deposited in GenBank under the accession number EU821598.     

Efficiencies of introduction of an oil-oxidizing <Emphasis Type="Italic">Dietzia maris</Emphasis> strain and stimulation of natural microbial communities in remediation of polluted soil

Two approaches to bioremediation of oil-polluted soils are compared: use of active degrader strain Dietzia maris AM3 and stimulation of natural microflora. Introduction of D. maris AM3 to soil freshly polluted with oil accelerated its remediation twofold within the first month in comparison with the stimulation. After three months, the purification degrees were approximately equal. By the end of bioremediation, the soil with the introduced strain had higher dehydrogenase and catalase activities. In soil with aged pollution, introduced strain D. maris AM3 did not affect the rate of oil product degradation, and no significant differences between the two bioremediation methods were detected in purification degree and biological activity of soil after three months.     

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent‐cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100‐fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1 × 10⁴ or more transformants per μg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0·25 mol l^?1 sucrose and 0·8 μg ml^?1 penicillin G, followed by treatment with 600 U ml^?1 lysozyme and electroporation at a field strength of 10 kV cm^?1. When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.     

Verrucosispora maris sp. nov., a novel deep-sea actinomycete isolated from a marine sediment which produces abyssomicins

Verrucosispora isolate AB-18-032^T, the abyssomicin- and proximicin-producing actinomycete, has chemotaxonomic and morphological properties consistent with its classification in the genus Verrucosispora. The organism formed a distinct phyletic line in the Verrucosispora 16S rRNA gene tree sharing similarities of 99.7%, 98.7% and 98.9% with Verrucosispora gifhornensis DSM 44337^T, Verrucosispora lutea YIM 013^T and Verrucosispora sediminis MS 426^T, respectively. It was readily distinguished from the two latter species using a range of phenotypic features and from V. gifhornensis DSM 44337^T, its nearest phylogenetic neighbor, by a DNA G+C content of 65.5 mol% obtained by thermal denaturation and fluorometry and DNA:DNA relatedness values of 64.0% and 65.0% using renaturation and fluorometric methods, respectively. It is apparent from the combined genotypic and phenotypic data that strain AB-18-032^T should be classified in the genus Verrucosispora as a new species. The name Verrucosispora maris sp. nov. is proposed for this taxon with isolate AB-18-032^T (= DSM 45365^T = NRRL B-24793^T) as the type strain.     

Agrobacterium-mediated Transformation of Brassica juncea with a Cyanobacterial (Synechocystis PCC6803) Delta-6 Desaturase Gene Leads to Production of Gamma-linolenic Acid

Genetic manipulation of the oil-yielding crop plants for better oil quality through biotechnological methods is an important aspect of crop improvement. Due to the inherent absence of the Δ⁶-desaturase (d6D) function, Brassica juncea, an oil-yielding crop plant, is unable to synthesize γ-linolenic acid (GLA), a nutritionally important fatty acid although the crop plant synthesizes the precursor fatty acids required for GLA production. Cyanobacterial d6D introduces carbon–carbon double bond onto linoleic acid (C18:2) and α-linolenic acid (C18:3) by desaturation processes for production of GLA and octadecatetraenoic acid (OTA) respectively. In the present investigation, d6D coding sequence from Synechocystis sp. PCC6803 was cloned by polymerase chain reaction and introduced into B. juncea through Agrobacterium-mediated transformation technique. Both cytosolic as well as seed-specific expression of d6D were attempted. The transformed plants show production of GLA and OTA in contrast to their absence in the untransformed control plants adducing evidence for introgression and functional expression of the cyanobacterial d6D gene in B. juncea.     

Construction of a new host-vector system inArthrobacter sp. and cloning of the lipase gene

Arthrobacter sp. strain MIS38 was transformed with a shuttle vector containing the kanamycin resistant genekan (derived from Tn5) by an electroporation method. This shuttle vector is fromBrevibacterium lactofermentum andEscherichia coli, pULRS8: - The following optimal condition of electroporation was determined. A square wave pulse of 1 kV/cm electric field strength for 0.5 ms duration yielded 3 × 10⁵ transformants/,g plasmid DNA. The number of transformants increased with the amount of DNA over the range 0.01-5 g. This host-vector system was then used successfully to clone and express a lipase gene fromArthrobacter sp. strain MIS38 into bothArthrobacter sp. MIS38 and E. coli JM109.     

Aerobic heterotrophic bacteria and petroleum-utilizing bacteria from cow dung and poultry manure

In this study, enumeration and identification of total aerobic heterotrophic bacteria and petroleum-utilizing bacteria as well as the degradative potential of petroleum-utilizing bacterial isolates were carried out. The average counts of total aerobic heterotrophic bacteria in cow dung and poultry manure were 74.25 × 10⁵ c.f.u. g⁻¹ and 138.75 × 10⁵ c.f.u. g⁻¹ respectively. Acinetobacter sp, Bacillus sp, Pseudomonas sp, and Serratia spp. occurred as aerobic heterotrophs in both cow dung and poultry manure. However, Alcaligenes spp. occurred only in cow dung while, Flavobacterium sp, Klebsiella sp, Micrococcus sp, and Nocardia spp. occurred only in poultry manure as aerobic heterotrophs. The average counts of petroleum-utilizing bacteria in cow dung and poultry manure were 9.25 × 10⁵ c.f.u. g⁻¹ and 17.25 × 10⁵ c.f.u. g⁻¹ respectively. Pseudomonas spp. occurred as petroleum utilizer in both cow dung and poultry manure. However, Bacillus spp. occurred only in cow dung while Acinetobacter spp. and Micrococcus spp. occurred only in poultry manure as petroleum utilizers. Relative abundance of petroleum utilizers in total aerobic heterotrophs ranged from 6.38% to 20.00% for cow dung and from 9.38% to 17.29% for poultry manure. Introduction of pure cultures of petroleum-utilizing bacteria from cow dung and poultry manure into sterile oil-polluted soil revealed oil degradation in one week period.     

Improvement of efficiency of genetic transformation for <Emphasis Type="Italic">Dunaliella salina</Emphasis> by glass beads method

Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10² transformants/μg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.     

Isolation and characterization of a novel strain of genus <Emphasis Type="Italic">Dietzia</Emphasis> capable of multiple-extreme resistance

An ultraviolet (UV) radiation resistant gram-positive bacterium, Dietzia sp. MG4 strain, was isolated from the Sirch Hot Spring (Kerman, Iran), then it was identified on the basis of morphological and biochemical characteristics, and 16S rRNA gene sequencing. The effects of temperature, pH, desiccation, different percentage of NaCl, hydrogen peroxide (H₂O₂), mitomycin C (MMC) and high levels of radiation on viability or growth rate of MG4 strain were investigated. Also heavy metal tolerance of MG4 strain was assayed. 16S rDNA sequence of the isolate exhibited 99.69% similarity with Dietzia sp. and this result was confirmed by phylogenetic analysis. Viability of this strain was obtained D₉₁ according to D index after exposure to 25 J/cm² UV radiation dose, and D₃₀ after desiccation stress (for 28 days) using flow cytometery. The D₁₀ value for a microorganism is defined as the stress dose necessary to provide 10% survivors. Therefore, this strain showed high resistance to UV-C radiation and moderate resistance to desiccation. Optimal growth of MG4 strain was observed at pH 9, temperature of 30°C and 5% (w/v) NaCl. Isolated Dietzia was resisted up to 3 mM of nickel and 0.2 mM of mercury ions. Also this strain could tolerate 1–4% (v/v) H₂O₂ and 8 µg/mL of MMC as oxidant agents. To the best of our knowledge, this is the first study on multiple extreme resistant Dietzia sp. MG4 strain.     

Improved conditions for the transformation by electroporation of the extracellular polysaccharide-producing methylotroph Methylobacillus sp.

The transformation efficiency of Methylobacillus sp. strain 12S, using electroporation, was unaffected by the growth phase of the cells but competent cells grown at 21 °C had a 1.9 × 10³ times higher transformation efficiency than those grown at 30 °C. Heat shock treatment further increased the transformation efficiency up to 7 times.     

Molecular detection and characterization of Theileria sp. from hedgehogs (Paraechinus aethiopicus) in Saudi Arabia

In this study, we conducted molecular detection and characterization of piroplasms that infect the Ethiopian or desert hedgehogs (Paraechinus aethiopicus) in Saudi Arabia. Blood samples from 112 (68 males and 44 females) desert hedgehogs from Unaizah, Central Saudi Arabia were screened for Theileria/Babesia DNA using the polymerase chain reaction (PCR) employing specific primers amplifying the partial 18S small subunit rRNA gene. Theileria DNA was detected in 51 samples (45·5%), giving a prevalence of 45·5%. Theileria DNA was found in 33 (48·5%) males and 18 (40·9%) females, and there was no significant difference (P > 0·05) in the prevalence between males and females. Similarly, there was no significant difference (P > 0·05) in the prevalence between juveniles (40%) and adults (46·7%). There was a significant difference in the prevalence of Theileria in hedgehogs collected from May to September and the period from October to April (P = 0·003). Four haplotypes of Theileria sp. in hedgehogs were detected and designated as H1–H4. H1 was the predominant haplotype and found in 80·8% of the positive individuals. Partial sequences of the 18S rRNA of Theileria sp. from hedgehogs grouped with Theileria spp. that are benign. This study is the first report of the occurrence of Theileria spp. in Saudi Arabian desert hedgehogs.     

Molecular Phylogeny and Surface Morphology of Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp. (Gregarinasina,Apicomplexa) Isolated from a Deep Sea Oradarea sp. (Amphipoda) in the West Pacific

In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper‐Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. Thiriotia hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this latter new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans.     

Transformation of <Emphasis Type="Italic">Platymonas</Emphasis> (<Emphasis Type="Italic">Tetraselmis</Emphasis>) <Emphasis Type="Italic">subcordiformis</Emphasis> (Prasinophyceae,Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25°C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10⁷ cell ml⁻¹. The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10⁻⁵, and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.     

Characterization of bacteria of the genus Dietzia: an updated review

The aim of this study was to characterize several aspects of species of the genus Dietzia, such as current taxonomic placement, morphological and growth characteristics, biochemical reactions, cellular lipid and fatty acid composition, the amino acids and sugars of whole-cell hydrolysates and the respiratory quinone system, and genomic guanine and cytosine (G + C) content. The species chosen for study were D. aerolata, D. alimentaria, D. aurantiaca, D. cerdiciphylli, D. cinnamea, D. kunjamensis, D. lutea, D. maris, D. natronolimnaea, D. papillomatosis, D. psychralcaliphila, D. schimae, and D. timorensis. The colony morphology study revealed that the colonies were small, smooth, circular and convex. Nitrate reduction, H₂S production, hydrolysis of urea, starch, and Tween 80, and the Voges–Proskauer and methyl red tests were performed for biochemical differentiation of the various Dietzia strains. Optimum growth temperature and pH for the different strains were 25–30 °C and 7–8, respectively. Among the strains studied, D. timorensis ID05-A0528^T had the lowest tolerance level to NaCl (7 %). This strain was also able to utilize a wide range of compounds as the sole carbon source. Short-chain mycolic acids were present in these bacteria. The cell wall contained meso-diaminopimelic acid, arabinose, and galactose; the glycan moiety of the cell wall contained acetyl residues. The major menaquinone was MK-8 (H₂). The G + C contents of the DNA ranged from 64.7 (D. alimentaria 72^T) to 73 mol?% (D. maris DSM 43672^T). The most important phospholipids in these strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, and phosphatidylethanolamine.