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.     

Cadmium biosorption by <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic"> circulans</Emphasis> strain EB1

Summary A heavy metal resistant bacterium, Bacillus circulans strain EB1 showed a high cadmium biosorption capacity coupled with a high tolerance to this metal when grown in its presence. Bacillus circulans EB1 cells grown in the presence of 28.1 mg cadmium/l were capable of removing cadmium with a specific biosorption capacity of 5.8 mg Cd/g dry wt biomass in the first 8 h. When the cells were pre-conditioned with low concentrations of cadmium in pre-grown medium, the uptake was increased to 6.7 mg Cd/g dry wt biomass. The maximum uptake of␣cadmium was during mid-logarithmic phase of growth. The resting cells (both wet and dry) of EB1 were also able to biosorb cadmium. Specific biosorption capacities of wet and dry biomass were 9.8 and 26.5 mg Cd/g dry wt biomass, respectively. Maximum cadmium removals by both wet and dry cells were at pH 7.0. The results showed that the cadmium removal capacity of resting cells was markedly higher than that of growing cells. Since both growing and resting cells had a high biosorption capacity for cadmium, EB1 cells could serve as an excellent biosorbent for removal of cadmium from natural environments.     

<Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mayteni</Emphasis> sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

An actinomycete strain, designated YIM 60475^T, was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained ll-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA–DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475^T is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475^T (=CCTCC AA 207005^T = KCTC 19383^T). Hua-Hong Chen and Sheng Qin contributed equally to this work.     

Extracellular phycoerythrin-like protein released by freshwater cyanobacteria <Emphasis Type="Italic">Oscillatoria</Emphasis> and <Emphasis Type="Italic">Scytonema</Emphasis> sp.

During growth of the freshwater cyanobacteria, Oscillatoria sp. BTCC/A0004, and Scytonema sp. TISTR 8208, a pink pigment is released into the growth medium. The pigment from each source had a molecular weight of approximately 250 kDa and had adsorption maxima at 560 and 620 nm. These results suggest that pink pigment is a phycoerythrin-like protein. It inhibited the growth of green algae, Chlorella fusca and Chlamydomonas reinhardtii, but not other cyanobacteria or true bacteria. The concentration at which growth inhibition 50% occurred was 0.5, 6 and more than 10 mg ml⁻¹, respectively.     

Feather degradation by a new keratinolytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. MS-2

Seventy different actinomycete isolates were evaluated for their ability to produce keratinase using a keratin-salt agar medium containing ball-milled feather as substrate. A novel feather-degrading isolate obtained from marine sediment produced the highest keratinolytic activity when cultured on broth containing whole feather as a primary source of carbon, nitrogen and energy. Based on phenotypic characterization and analysis of 16S rDNA sequencing the isolate was identified as a Streptomyces sp. MS-2. Maximum keratinase activity (11.2 U/mg protein) was achieved when cells were grown on mineral salt liquid medium containing 1% whole chicken feather adjusted to pH 8 and incubated at 35°C for 72 h at 150 rpm. Reduction of disulphide bridges was also detected, increasing with incubation time. Feather degradation led to an increase in free amino acids such as alanine, leucine, valine and isoleucine. Moreover, methionine and phenylalanine were also produced as microbial metabolites.     

Rapid Affinity Purification Processes for Cyclodextrin Glycosyltransferase from <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">circulans</Emphasis>

Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM α-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.     

Effective biotic elicitation of <Emphasis Type="Italic">Ruta graveolens</Emphasis> L. shoot cultures by lysates from <Emphasis Type="Italic">Pectobacterium atrosepticum</Emphasis> and <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 μg g⁻¹ dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 μg g⁻¹ dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 μg g⁻¹ dry wt With lysates of the Pectobacterium atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of γ-fagarine, skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 μg g⁻¹ dry wt, respectively.     

Study of redox potential in cytochrome <Emphasis Type="Italic">c</Emphasis> covalently bound to terminal oxidase of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> FTU

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E(m)(app)) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction.     

Study of biodiversity of <Emphasis Type="Italic">Klebsiella </Emphasis>sp.

Microorganisms provide the largest gamut of genetic and physiological diversity in nature. The determination of the biodiversity of microbes is necessary for the exploitation of their inestimable population, which remains unexplored in the environment. In our study, we have isolated 20 different Klebsiella sp. from contaminated soil and effluent treatment plants and determined their genetic diversity using molecular tools. The phylogenetic result based on 16S rDNA sequencing, shows genetic variation among the strains, which was further confirmed by Randomly Amplified Polymorphic DNA (RAPD) analysis. The occurrence of a broad spectrum of Klebsiella sp. variants in polluted and stress environments portrays the ability of this genus to survive abundantly under stress conditions.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Effect of temperature on the life history of <Emphasis Type="Italic">Eretmocerus</Emphasis> sp. nr. <Emphasis Type="Italic">furuhashii</Emphasis>, a parasitoid of <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Eretmocerus sp. nr. furuhashii (Hymenoptera: Aphelinidae) is an indigenous parasitoid of Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae) from southern China; the effects of constant temperatures on the life history of E. sp. nr. furuhashii were examined in the laboratory. The developmental period ranged from 39.2 days at 20°C to 12.40 days at 32°C. A total of 263.4 degree-days were required to complete development with a lower developmental threshold temperature of 11.1°C. Of the eggs produced, 59.3% completed development at 20°C with completion increasing to 71.5% at 26°C. Adult female longevity was 10.8 days at 20°C and 5.2 days at 32°C while the mean daily offspring reproduced per female was highest at 29°C with 5.9 offspring. Adult oviposition peaked three days after emergence at 26, 29 and 32°C, and four days post-emergence at 20°C and 23°C. The total numbers of offspring produced per female ranged from 25.7 individuals at 32°C to 41.1 individuals at 20°C. The sex ratio had a female bias and ranged from 0.72 at 17°C to 0.51 at 35°C. The intrinsic rate of increase was 0.1727 at 29°C followed with 0.1606 at 32°C. Results indicated that E. sp. nr. furuhashii reaches its maximum biological potential at temperatures ranging from 26°C to 32°C.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.     

Molecular identification of alkaliphilic and halotolerant strain<Emphasis Type="Italic"> Bacillus</Emphasis> sp. FTU as<Emphasis Type="Italic"> Bacillus pseudofirmus</Emphasis> FTU

The systematic position of the alkaliphilic and halotolerant strain Bacillus sp. FTU was refined in view of the comprehensive taxonomic revision of the group of alkaliphilic and alkalitolerant Bacillus strains. Sequence analysis of almost the entire 16S rRNA gene of Bacillus sp. FTU revealed 99.8% homology with two Bacillus pseudofirmus strains. Subsequent DNA-DNA hybridization analysis confirmed the close relationship of Bacillus sp. FTU with the type strain of B. pseudofirmus (the level of homology reached 86%). Results of physiological and biochemical characterizations relevant for the group clearly underlined the positioning of strain FTU within this species. It is therefore concluded that Bacillus sp. FTU represents a strain of the alkaliphilic species B. pseudofirmus and is to be renamed as B. pseudofirmus FTU. The phylogeny of different Bacillus species is discussed using N-terminal sequence homologies of some caa (3)-type oxidase subunits.     

Over-expression of <Emphasis Type="Italic">CONSTANS-LIKE 5</Emphasis> can induce flowering in short-day grown <Emphasis Type="Italic">Arabidopsis</Emphasis>

To ensure that the initiation of flowering occurs at the correct time of year, plants need to integrate a diverse range of external and internal signals. In Arabidopsis, the photoperiodic flowering pathway is controlled by a set of regulators that include CONSTANS (CO). In addition, Arabidopsis plants also have a family of genes with homologies to CO known as CO-LIKE (COL) about which relatively little is known. In this paper, we describe the regulation and interactions of a novel member of the family, COL5. The expression of COL5 is under circadian and diurnal regulation, but COL5 itself does not appear to affect circadian rhythms. COL5, like CO, is regulated by GIGANTEA. Furthermore, COL5 is expressed in the vascular tissue. Using COL5 over-expressing lines we show that, under short days, constitutive expression of COL5 affects flowering time and the expression of the floral integrator genes, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO 1. Constitutive expression of COL5 partially suppresses the late flowering phenotype of the co-mutant plants. However, plants with loss of COL5 function do not show altered flowering. Taken together, our results suggest that COL5 has COL activity, but may either not have a role in regulating flowering in wild-type plants or may act redundantly with other flowering regulators. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mineralization of chlorpyrifos by co-culture of <Emphasis Type="Italic">Serratia</Emphasis> and <Emphasis Type="Italic">Trichosporon</Emphasis> spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).     

Allelopathic impact of artificially applied coumarin on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis>

Watermelon production is threatened by fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON) in continuous cultivation system. Some elements, mainly allelochemicals, released from living roots or decayed plants might be associated with the disease. The purpose of this work was to evaluate the possible impact of coumarin, one kind of watermelon allelochemical, on FON. Furthermore, possible new mechanisms might be investigated during the ecological interactions of plant-microbe. Results showed that coumarin strongly inhibited growth of FON leading to a decrease in its biomass, dry weight of mycelia of FON in a liquid culture. The dry weight was decreased by 62.9% compared with control. The hyphal growth of FON on plates was stopped at high (>400 mg l⁻¹) concentrations of coumarin. At 320 mg l⁻¹, sporulation and enzyme activities of FON were also severely suppressed by coumarin. The yield of conidia, and the activities of proteinase, cellulase, and amylase were reduced by 98.9%, 79.7%, 29.8% and 15.9% respectively. However, conidial germination and mycotoxin (MT) production of FON were greatly stimulated, being increased by 55.7% and 14.9 fold at 320 mg l⁻¹ respectively. We conclude that coumarin acted as an allelochemical substance to inhibit growth and pathogenic enzyme activities of FON but to stimulate mycotoxin production and conidial germination. It was suggested that coumarin acted as a signal transduction element bridging plant and pathogen in the process of plant-microbe interactions.     

Energetics of alkalophilic representatives of the genus <Emphasis Type="Italic">Bacillus</Emphasis>

Cytochrome and lipid composition of membranes is considered as the attributes required for adaptation of the alkalophiles to alkaline conditions. Respiratory chains of alkalophilic representatives of the genus Bacillus are discussed. Special attention is paid to the features of the Na⁺-cycle of these bacteria and to the features determining halo- and alkalotolerant phenotype, which have been reported due to recent achievements in genomics.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 171–176.Original Russian Text Copyright © 2005 by Muntyan, Popova, Bloch, Skripnikova, Ustiyan.This revised version was published online in April 2005 with corrections to the post codes.     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.     

<Emphasis Type="Italic">In vitro</Emphasis> DNA-binding properties of a manganese response regulator (ManR) from <Emphasis Type="Italic">Anabaena</Emphasis> sp.

ManR of Anabaena sp. PCC 7120 is a manganese response regulator. Two ManR molecules bind to the specific DNA sequences at the same time, which was demonstrated by our previous results. From size exclusion chromatography, ManR exits as monomer in solution. Therefore, cooperative interactions of ManR–ManR play a role in DNA binding of the ManR, suggesting that ManR molecules bind co-operatively to DNA. When serial deletions of N-terminal of the ManR were also carried out the mutant proteins, ManRC111, ManRC130 and ManRC158, had completely lost the in DNA binding activity. Mutants ManRC 196, ManRC206, ManRC221 and ManRC230, however, could specifically bind to DNA, indicating that the amino acid residues between Val₁₆ and Ile₇₈ of the N-terminal of ManR are necessary for the DNA binding activity of C-terminal domain.Revisions requested 20 Ocotober 2004/15 November 2004; Revisions received 10 November/13 December 2004