Characterisation of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Ochrobactrum</Emphasis> sp. isolated from volcanic soil

Soil bacteria may have properties of plant growth promotion but not be sufficiently beneficial for plants under stress conditions. This challenge has led researchers to extend their searches into extreme environments for potential soil bacteria with multiple plant beneficial traits as well as abiotic stress tolerance abilities. In the current study, an attempt was made to evaluate soil bacteria from an extreme environment, volcano soils, based on plant growth promoting and abiotic stress mitigating characteristics. The screening led to the isolation of eight (NBRISH4, NBRISH6, NBRISH10, NBRISH11, NBRISH13, NBRISH14, NBRISH16 and NBRISH26) bacterial isolates capable of withstanding stresses, namely temperature (up to 45 °C), salt (up to 2 M NaCl) and drought (up to 60% Poly Ethylene Glycol 6000) in vitro. Further, the selected isolates were notable for their in vitro temporal performance with regards to survival (in terms of colony count), phosphate solubilisation, biofilm formation, auxin, alginate and exo-polysaccharide production abilities under abiotic stresses i.e. 40 °C temperature; 500 mM NaCl salt and drought (PEG) conditions. In vivo seed treatments of individual selected bacteria to maize plants resulted into significant enhancement in root and shoot length, root and shoot fresh and dry weight and number of leaves per plant. Overall, the plant growth promoting and abiotic stress tolerance ability was most evident for bacterial isolate NBRISH6 which was identified as an Ochrobactrum sp. using 16S rRNA based phylogenetic analysis.     

Salicylate degradation by a cold-adapted <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Pseudomonas sp. strain MC1 was characterized as a cold-adapted, naphthalene-degrading bacterium that is able to grow in a broad temperature range of 5–30°C. MC1 harbors a catabolic plasmid, designated pYIC1, which is almost identical to the archetypal NAH7 plasmid from the mesophilic bacterium Pseudomonas putida G7. On pYIC1, the catabolic genes for naphthalene degradation are clustered in two operons: nahAa-Ab-Ac-Ad-B-F-C-Q-E-D encoding the conversion of naphthalene to salicylate, and nahG-T-H-I-N-L-O-M-K-J encoding the conversion of salicylate through meta-cleavage pathway to pyruvate and acetyl CoA. NahH, the bona fide extradiol dioxygenase in MC1 salicylate metabolism, is thermolabile and is a cold-adapted enzyme. The thermal profiles of the NahH enzyme activities expressed in different hosts indicate the presence of a factor(s) or mechanism(s) to protect the thermolabile NahH enzyme (100% aa identity with MC1 counterpart) in G7. Overall, the results reported in the present work suggest that the thermolabile NahH might be a product of the cold-adaptation process of MC1 and thus contribute to the survival and growth ability of MC1 on salicylate and naphthalene in cold environments.     

Biodegradation of Ethametsulfuron-Methyl by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SW4 Isolated from Contaminated Soil

A soil bacterium SW4, capable of degrading the sulfonylurea herbicide ethametsulfuron-methyl (ESM), was isolated from the bottom soil of a herbicide factory. Based on physiological characteristics, biochemical tests and phylogenetic analysis of the 16S rRNA gene sequence, the strain was identified as a Pseudomonas sp. The total degradation of ESM in the medium containing glucose was up to 84.6% after 6 days of inoculation with SW4 strain. The inoculation of strain SW4 to soil treated with ESM resulted in a higher degradation rate than in noninoculated soil regardless of the soil sterilized or nonsterilized. Five metabolites of ESM degradation were analyzed by liquid chromatography/mass spectrometry. Based on the identified products, strain SW4 seemed to degrade ESM after two separate and different pathways: one leads to the cleavage of the sulfonylurea bridge, whereas the other to the dealkylation and opening of the triazine ring of ESM.     

Dearomatization of diesel oil using <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Objectives

To improve the quality of diesel fuel via removal of aromatic compounds using Pseudomonas sp.

Results

In the present study Pseudomonas sp. was able to remove 94% of fluorene, 59% of phenanthrene, 49% of anthracene, 52% of fluoranthene, 45% of pyrene and 75% carbazole present in diesel oil. Additionally, it also does not affect the aliphatic content of fuel thus maintaining the carbon backbone of the fuel.

Conclusions

Pseudomonas sp. is a potential biocatalyst that can be used in the refining industry.

     

<Emphasis Type="Italic">Kretzschmaria varians</Emphasis> sp. nov., <Emphasis Type="Italic">Xylaria coremiifera</Emphasis> sp. nov. and <Emphasis Type="Italic">Xylaria umbonata</Emphasis> sp. nov. from Costa Rica

Kretzschmaria varians, a species apparently related to K. micropus, is described as new. It is distinguished primarily by having asci with 2 to 8 ascospores with inconstant germination slit length and remains of synnemata on stromata and surrounding substrate. Xylaria coremiifera, described here as new, bears small fragile coremia on pulvinate stromata and the surrounding substrate. Asci often have fewer than 8 ascospores, most frequently 4. Xylaria umbonata, described here as new, produces perithecia around a central umbo that appears to be the remains of a synnema. Ascospores have long spiralling germination slits.     

<Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> associated bioactive bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.From Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 35–39.Original English Text Copyright © 2005 by Li Zheng, Xiaojun Yan, Jilin Xu, Haimin Chen, Wei Lin.This article was submitted by the authors in English.     

High production of laccase B from <Emphasis Type="Italic">Trametes</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The coding sequence of a laccase isozyme from Trametes sp. AH28-2 was cloned in pPIC9K vector and heterologously overexpressed in the yeast Pichia pastoris strain GS115. In the minimal medium containing 0.3 mM CuSO₄ and 0.6% alanine, the maximum yield of the recombinant laccase rLacB reached 32,000 U/l (1,012 U/mg), slightly higher than that of the native enzyme nLacB (∼30,000 U/l, 1,356 U/mg). The enzymatic properties of rLacB were different from those of nLacB as well. Regardless of the inferior thermal stability, rLacB had much better stability at both neutral and basic pH range compared to nLacB. In addition, the dye decolorization potential of rLacB was similar to that of nLacB.     

Control of pyrimidine nucleotide formation in <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fulva</Emphasis>

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 5′-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.     

<Emphasis Type="Italic">Cohnella ferri</Emphasis> sp. nov. A Novel Member of the Genus <Emphasis Type="Italic">Cohnella</Emphasis> Isolated from Haematite Ore

The taxonomic position of a Gram-positive, endo-spore forming bacterium isolated from a haematite ore sample was analyzed by a polyphasic approach. The strain designated as HIO-4^T matched most of the phenotypic and chemical characteristics of the genus Cohnella and represents a novel species. The sequence of the almost complete 16S rRNA (1489 bases) was compared with those of previously studied Cohnella type strains and confirmed that the strain belongs to the genus Cohnella. Strain HIO-4^T differs from all other species of Cohnella by at least 3.9% at the 16S rRNA level and the moderately related species are Cohnella phaseoli (96.1%) and Cohnella yongneupensis (96.1%), respectively. Predominant polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE); few unknown phospholipids, mannose containing lipid, aminophospholipid and aminophosphoglycolipids. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinctiveness of strain HIO-4^T with its phylogenetic relatives and suggest that the strain HIO-4^T should be recognized as a novel species, for which the name Cohnella ferri sp. nov. is proposed. The type strain is HIO-4^T (=MTCC 8365^T = JCM 16139^T)     

<Emphasis Type="Italic">Actimicrobium antarcticum</Emphasis> gen. nov., sp. nov., of the Family <Emphasis Type="Italic">Oxalobacteraceae</Emphasis>, Isolated from Antarctic Coastal Seawater

A Gram-negative, non-motile, catalase- and oxidase- positive, strictly aerobic, and short rod-shaped bacterium that was designated strain KOPRI 25157^T was isolated from coastal seawater sample in Antarctica. The temperature and pH ranges for growth on R2A agar were 10–20°C, and 5.0–10.0, respectively. Phylogenetic analyses of the 16S rRNA gene sequence of strain KOPRI 25157^T showed it to belong to the family Oxalobacteraceae of the class Betaproteobacteria, and it formed a distinct clade from other recognized members of the family. DNA G + C content was 65.9 mol%. Major ubiquinone was Q-8. Predominant cellular fatty acids were C_16:1 ω7c/15 iso 2OH (56.4%) and C_16:1 (30.5%). Major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and unknown lipid. On the basis of these data, it is proposed that strain KOPRI 25157^T is the representative of a novel genus, for which the name Actimicrobium gen. nov. is proposed in the family Oxalobacteraceae. The type strain for Actimicrobium antarcticum sp. nov. is KOPRI 25157^T (=JCM 16673^T=KCTC 23040^T).     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

Expression and Characterization of the TNT Nitroreductase of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pseudomonas sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V _max and K _m values of the enzyme for TNT were 12.6 μmol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and stop codons. Eight days was required for complete degradation of 0.5 mM TNT by the wild-type HK-6 strain, whereas the pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50% of the 0.5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial role in the TNT degradation pathway of the HK-6 strain.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Biodegradation of malachite green by strain <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K9 and cloning of the <Emphasis Type="Italic">tmr2</Emphasis> gene associated with an IS<Emphasis Type="Italic">Ppu12</Emphasis>

A bacterial strain K9 capable of degrading malachite green was isolated from the sludge of the wastewater treatment system of a chemical plant. It was identified preliminarily as Pseudomonas sp. Strain K9 was also able to degrade other triphenylmethane dyes, such as Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase, was cloned from strain K9, and functionally expressed in E. coli. A 5946-bp DNA fragment including the tmr2 gene was cloned from the genomic DNA of strain K9 by chromosome walking. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase) and orf1 (encoding a putative MerR family regulator), orf2 (encoding a CDF family heavy metal/H⁺ antiporter). This association was also found in another malachite green-degrading strain Pseudomonas sp. MDB-1, which indicated that the tmr2 gene might be a horizontally transferable gene.     

Cyanobactericidal effect of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. isolated from eutrophic lake on <Emphasis Type="Italic">Microcystis</Emphasis> sp

A bacterium, which was observed in all cultivations of Microcystis sp., was isolated and designated as Rhodococcus sp. KWR2. The growth of bloom-forming cyanobacteria, including four strains of Microcystis aeruginosa and Anabaena variabilis, was suppressed by up to 75–88% by 2% (v/v) culture broth of KWR2 after 5 days. But KWR2 did not inhibit eukaryotic algae, Chlorella vulgaris and Scenedesmus sp. An extracellular algicidal substance produced by KWR2 showed a cyanobactericidal activity of 94% and was water-soluble with a molecular weight of lower than 8 kDa.     

Multilocus phylogeny of <Emphasis Type="Italic">Clonostachys</Emphasis> subgenus <Emphasis Type="Italic">Bionectria</Emphasis> from Brazil and description of <Emphasis Type="Italic">Clonostachys chloroleuca</Emphasis> sp. nov.

Phylogenetic analyses based on protein-encoding gene exons and introns of ATP citrate lyase (ACL1), beta tubulin (TUB), the largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-α (TEF1) are used for inferring the existence of a new Clonostachys species from the Cerrado biome in Brazil, described here as C. chloroleuca. The species produces dimorphic, primary, and secondary conidiophores that form consistently greenish conidial masses on artificial media. It resembles therefore C. rosea f. catenulata although it differs from this species by less adpressed branches in the secondary conidiophores. The new species is also phylogenetically related to C. byssicola and C. rhizophaga. Our inventory suggests that C. byssicola, C. chloroleuca, C. pseudochroleuca, C. rhizophaga, C. rogersoniana, and C. rosea commonly occur in native and agriculturally used soils of the Cerrado and Amazon Forest. Using sequences available from two genome-sequenced strains employed as biological control agents, we confirm the identity of the European strain IK726 as C. rosea and identify strain 67-1 from China as C. chloroleuca.