Dichloromethane as the sole carbon source forHyphomicrobium sp. strain DM2 under denitrification conditions

Hyphomicrobium sp. strain DM2 was found to grow anaerobically in the presence of nitrate with methanol, formaldehyde, formate or dichloromethane. The estimated growth rate constants with methanol and dichloromethane under denitrification conditions were 0.04 h^–1 and 0.015 h^–1, respectively, which is twofold and fourfold lower than the rates of aerobic growth with these substrates. Slight accumulation of nitrite was observed in all cultures grown anaerobically with nitrate. Dichloromethane dehalogenase, the key enzyme in the utilization of this carbon source, was induced under denitrification conditions to the same specific activity level as under aerobic conditions. In a fed batch culture under denitrification conditionsHyphomicrobium sp. DM2 cumulatively degraded 35 mM dichloromethane within 24 days. This corresponds to a volumetric degradation rate of 5 mg dichloromethane/l·h and demonstrates that denitrificative degradation offers an attractive possibility for the development of anaerobic treatment systems to remove dichloromethane from contaminated groundwater.     

Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with μ_max of 0.094 h⁻¹ and S _m of 1,435 mg l⁻¹. Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.     

Dehalogenation of dichloromethane by cell extracts of hyphomicrobium DM2

A facultatively methylotrophic bacterium was isolated from enrichment cultures containing dichloromethane as the sole carbon source. It was identified as a Hyphomicrobium species. The organism grew exponentially in batch cultures with 10 mM dichloromethane at a specific growth rate of 0.07 h^-1. The release of Cl^- from dichloromethane and the disapperance of substrate paralleled growth. Resting dichloromethane-grown cells, in the presence of potassium sulphite as a trapping agent, converted cichloromethane methane quantitatively to formaldehyde. The conversion of dichloromethane to formaldehyde by cell extracts was stricly dependent on glutathione. Other thiols were inactive. Glutathione was not consumed in the course of the reaction. The specific activity of the enzymic dehalogenation of dichloromethane amounted to 3.8 mkat/kg protein in extracts of dichloromethane-grown cells and to less than 0.1 mkat/kg protein in extracts from cells grown on methanol.     

Mapping of some genes involved in C-1 metabolism in the facultative methylotroph Methylobacterium sp strain AM1 (Pseudomonas AM1)

R68.45 mediated mobilisation of the chromosome of Methylobacterium sp strain AM1 has been investigated. High frequencies of cotransfer of four genes required for C-1 metabolism with the genes coding for streptomycin, phosphonomycin and cycloserine resistance were demonstrated. A preliminary map of this region has been constructed on the basis of the results of three and four factor crosses showing that not all the C-1 genes are contiguous.Abbreviations Str streptomycin - Pho phosphonomycin - Cyc cycloserine - Tc tetracycline - Km kanamycin - Cb carbenicillin - Ade adenine - Thi thiamine - Met methionine     

Microbiological analysis of cryopegs from the Varandei Peninsula,Barents Sea

The paper deals with the microbiological characterization of water-saturated horizons in permafrost soils (cryopegs) found on the Varandei Peninsula (Barents Sea coast), 4–20 m deep. The total quantity of bacteria in the water of cryopegs was 3.5 × 10⁸ cells/ml. The population of cultivated aerobic heterotrophic bacteria was 3–4 × 10⁷ cells/ml and the number of anaerobic heterotrophic bacteria varied from 10² to 10⁵ cells/ml depending on cultivation temperature and salinity. Sulfate-reducing bacteria and methanogenic archaea were found as hundreds and tens of cells per ml of water, respectively. A pure culture of a sulfate-reducing strain B15 was isolated from borehole 21 and characterized. Phylogenetic analysis has shown that the new bacterium is a member of the genus Desulfovibrio with Desulfovibrio mexicanus as its closest relative (96.5% similarity). However, the significant phenotypic differences suggest that strain B15 is a new species of sulfate-reducing bacteria.     

Identification and functional characterisation of genes and corresponding enzymes involved in carnitine metabolism of <Emphasis Type="Italic">Proteus</Emphasis> sp.

Enzymes involved in carnitine metabolism of Proteus sp. are encoded by the cai genes organised as the caiTABCDEF operon. The complete operon could be sequenced from the genomic DNA of Proteus sp. Amino acid sequence similarities and/or enzymatic analysis confirmed the function assigned to each protein involved in carnitine metabolism. CaiT was suggested to be an integral membrane protein responsible for the transport of betaines. The caiA gene product was shown to be a crotonobetainyl-CoA reductase catalysing the irreversible reduction of crotonobetainyl-CoA to -butyrobetainyl-CoA. CaiB and CaiD were identified to be the two components of the crotonobetaine hydrating system, already described. CaiB and caiD were cloned and expressed in Escherichia coli. After purification of both proteins, their individual enzymatic functions were solved. CaiB acts as betainyl-CoA transferase specific for carnitine, crotonobetaine, -butyrobetaine and its CoA derivatives. Transferase reaction proceeds, following a sequential bisubstrate mechanism. CaiD was identified to be a crotonobetainyl-CoA hydratase belonging to the crotononase superfamily. Because of amino acid sequence similarities, CaiC was suggested to be a betainyl-CoA ligase. Taken together, these results show that the metabolism of carnitine and crotonobetaine in Proteus sp. proceeds at the CoA level.     

Bacteriochlorophyll c monomers,dimers, and higher aggregates in dichloromethane,chloroform, and carbon tetrachloride

Bacteriochlorophyll c in vivo is a mixture of at least 5 homologs, all of which form aggregates in CH₂Cl₂, CHCl₃ and CCl₄. Three homologs exist mainly in the 2-R-(1-hydroxyethyl) configuration, whereas the other two homologs, 4-isobutyl-5-ethyl and 4-isobutyl-5-methyl farnesyl bacteriochlorophyll c, exist mainly in the 2-S-(1-hydroxyethyl) configuration (Smith KM, Craig GW, Kehres LA and Pfennig N (1983) J. Chromatograph. 281: 209–223). In CCl₄ the S-homologs form an aggregate of 2–3 molecules whose absorption (747 nm maximum) and circular dichroism spectra resemble those of the chlorosome. In CH₂Cl₂, CHCl₃ and CCl₄ the 4-n-propyl homolog (R-configuration) forms dimers absorbing at ca. 680 nm and higher aggregates absorbing at 705–710 nm. In CCl₄ the dimerization constant is approx. 10 µM^–1 (1000 times that for chlorophyll a). The difference between the types of aggregates formed by the 4-n-propyl and 4-isobutyl homologs is attributed to the difference between the R- and S-configurations of the 2-(1-hydroxyethyl) groups in each chlorophyll.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - EEF 4-ethyl-5-ethyl farnesyl - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - MEF 4-methyl-5-ethyl farnesyl - PEP 4-n-propyl-5-ethyl farnesyl     

The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts

Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication cost was partially covered by the organisers of this meeting.     

Review of the genusBembras Cuvier, 1829 (Scorpaeniformes: Bembridae) with description of three new species collected from Australia and Indonesia

The bembrid genusBembras Cuvier is reviewed. Five species,B. japonica Cuvier,B. adenensis Imamura & Knapp and three undescribed species, were assigned to the genus. Type species of the genus,Bembras japonica is redescribed on the basis of 36 specimens including the holotype, and three new species,B. macrolepis, B. longipinnis andB. megacephala, previously misidentified asB. japonicus, are also described on the basis of specimens collected from Australia and Indonesia.Bembras macrolepis differs from its congeners by having large body scales, a long pectoral fin with 17–19 rays and a dark blotch on slightly upper portion to middle of margin, 14–15 anal-fin rays, small head and orbit, and caudal fin with a broad vertical dark band near posterior margin.Bembras longipinnis is distinguished from other members of the genus by having a slightly long pectoral fin with 17–19 rays and lacking a small black blotch near tip of upper rays, caudal fin with a large dark spot most intense in lower lobe, 1–2 gill rakers on upper gill arch, 13–14 anal-fin rays, slightly elong ated head and small orbit.Bembras megacephala is characterized by the following combination of characters: caudal fin with several irregular narrow vertical dark bands, small orbit, pectoral fin with 19–20 rays and lacking a small black blotch near tip of upper rays, head elongate, 2–4 gill rakers on upper gill arch, 15 anal-fin rays and small body scales. A key separating the five species ofBembras is given.     

Mapping multiple disease resistance genes using a barley mapping population evaluated in Peru, Mexico, and the USA

We used a well-characterized barley mapping population (BCD 47 × Baronesse) to determine if barley stripe rust (BSR) resistance quantitative trait loci (QTL) mapped in Mexico and the USA were effective against a reported new race in Peru. Essentially the same resistance QTL were detected using data from each of the three environments, indicating that these resistance alleles are effective against the spectrum of naturally occurring races at these sites. In addition to the mapping population, we evaluated a germplasm array consisting of lines with different numbers of mapped BSR resistance alleles. A higher BSR disease severity on CI10587, which has a single qualitative resistance gene, in Peru versus Mexico suggests there are differences in pathogen virulence between the two locations. Confirmation of a new race in Peru will require characterization using a standard set of differentials, an experiment that is underway. The highest levels of resistance in Peru were observed when the qualitative resistance gene was pyramided with quantitative resistance alleles. We also used the mapping population to locate QTL conferring resistance to barley leaf rust and barley powdery mildew. For mildew, we identified resistance QTL under field conditions in Peru that are distinct from the Mla resistance that we mapped using specific isolates under controlled conditions. These results demonstrate the long-term utility of a reference mapping population and a well-characterized germplasm array for locating and validating genes conferring quantitative and qualitative resistance to multiple pathogens.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Four new species of the genusSporobolomyces isolated from leaves in Thailand

Thirteen undescribed strains of ballistoconidium-forming yeasts, isolated from leaves collected in the suburbs and along the southeast seacoast of Bangkok, Thailand, were divided into four different groups in the genusSporobolomyces on the basis of morphological, physiological, biochemical, and chemotaxonomical characteristics, and analyses of the sequences of 18S rDNA and internal transcribed spacer regions. DNA-DNA reassociation experiments with related species revealed that these four groups were four new distinct species.Sporobolomyces nylandii sp. nov.,S. poonsookiae sp. nov.,S. blumeae sp. nov. andS. vermiculatus sp. nov. are proposed for these strains.     

Cloning and expression of a haloacid dehalogenase fromPseudomonas sp. strain 19 S

A dehalogenase gene specifying the utilization of a variety of haloacids byPseudomonas sp. Strain 19S has been cloned and expressed inE. coli. Our cloning strategy employed specific amplification of a fragment homologous toPseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digestedPseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified inE. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.Abbreviations 2-CPA 2-chloropropionate - MCA monochloro acetate - IPTG isopropyl-1-thio--D-galactoside - NBT nitroblue tetrazolium salt - PCR polymerase chain reaction - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside - X-phosphate 5-bromo-4-chloro-3-indolyl phosphate     

Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants

In a taxonomic study on the ascomycetous yeasts isolated from plant materials collected in tropical forests in Yunnan and Hainan Provinces, southern China, four strains isolated from tree sap (YJ2E(T)) and flowers (YF9E(T), YWZH3C(T) and YYF2A(T)) were revealed to represent four undescribed yeast species. Molecular phylogenetic analysis based on the large subunit (26S) rRNA gene D1/D2 domain sequences showed that strain YJ2E(T) was located in a clade together with Candida haemulonii and C. pseudohaemulonii. Strain YF9E(T) was most closely related to C. azyma and strain YWZH3C(T) to C. sorbophila and C. spandovensis. Strain YYF2A(T) was clustered in a clade containing small-spored Metschnikowia species and related anamorphic Candida species. The new strains differed from their closely related described species by more than 10% mismatches in the D1/D2 domain. No sexual states were observed for the four strains on various sporulation media. The new species are therefore assigned to the genus Candida and described as Candida alocasiicola sp. nov. (type strain, YF9E(T) = AS 2.3484(T) = CBS 10702(T)), Candida hainanensis sp. nov. (type strain, YYF2A(T) = AS 2.3478(T) = CBS 10696(T)), Candida heveicola sp. nov. (type strain, YJ2E(T) = AS 2.3483(T) = CBS 10701(T)) and Candida musiphila sp. nov. (type strain, YWZH3C(T) = AS 2.3479(T) = CBS 10697(T)).     

Monomers,dimers, and tetramers of 4-n-propyl-5-ethyl farnesyl bacteriochlorophyll c in dichloromethane and carbon tetrachloride

Absorption (ABS) and circular dichroism (CD) spectra were recorded for 6 concentrations (2.0–290 M) of bacteriochlorophyll (BChl) c in each solvent. Monomer spectra were obtained by adding methanol (1:200) to each sample. The monomer showed an ABS peak and a CD trough at 664 nm in CH₂Cl₂ (ABS peak at 665 nm in CCl₄). Dimer-plus-monomer spectra were obtained by subtracting high concentration (e.g., 290 M) spectra appropriately scaled from lower concentration (e.g., 26 M) spectra. Pure dimer spectra were then obtained by subtracting monomer spectra appropriately scaled from dimer-plus-monomer spectra. The dimer showed an ABS peak at 679 nm in both CH₂Cl₂ and CCl₄ and a CD trough at ca. 670 nm in CH₂Cl₂. The optical properties of the dimer do not agree with the model for bacteriochlorophyllide d [Smith KM, Bobe FW, Goff DA and Abraham RJ (1986) J Am Chem Soc 108: 1111–1120]. Higher aggregate spectra were obtained by subtracting appropriately scaled monomer and dimer spectra from high concentration (e.g., 290 M) spectra. The aggregate showed ABS shoulders at ca. 636 and 678 nm with a peak at 702 nm in CH₂Cl₂ and at 708 nm in CCl₄; the CD spectrum in either solvent showed peaks at 638 and 679 nm with troughs at 658 and ca. 710 nm. These spectra are consistent with an excitonic interaction between 4 chromophores in the aggregate. Each of the 12 original ABS spectra was deconvoluted in terms of the appropriate monomer, dimer and aggregate spectra, and the concentrations of each component were determined. Plots of log aggregate concentration vs. log dimer concentration lay on or near a line of slope 1.9 for CH₂Cl₂ and on or near a line of slope 2.1 for CCl₄. The aggregate was thus shown to be a tetramer. The theoretical relationship between dimers and monomers (slope 2.0) was not observed in all cases.Abbreviations ABS absorbance - BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - PEF 4-n-propyl-5-ethyl farnesyl     

Organic matter fractions by SP-MAS C NMR and microbial communities involved in the suppression of Fusarium wilt in organic growth media

Fusarium wilt is an economically important disease in carnation and tomato plants. The use of suppressive plant growth media has become an alternative method for plant disease control due to the lack of effective chemical control measures. Plant disease suppressiveness is sustained only in plant growth media with an adequate organic matter (OM) composition. Carbohydrate polymers are the most important sources of carbon nutrient for microbial community in these media, mainly consisting of cellulose and hemicellulose. This determines microbial activity, biomass and selects microbial communities in plant growth media, which are reported factors associated with Fusarium wilt suppressiveness.This work determined OM carbon functional groups using Single Pulse Magic Angle Spinning ¹³C-Nuclear Magnetic Resonance (SP-MAS ¹³C-NMR) in three plant growth media with different suppressiveness levels to Fusarium wilt in two crops, carnation and tomato. We propose that the critical role of OM to sustain naturally occurring suppressiveness in those media is not related with cellulose reserve. This could be explained because cellulose protected by lignin encrustation is not available to microbial degradation, meaning that cellulose availability is critical to sustenance of microorganism-mediated biological control. However, the hemicellulose relative abundance (peak 175 ppm) was associated to Fusarium wilt suppression level in plant growth media studied.Carbon source availability in OM was related to microbial biomass and econutritional group population densities involved in biocontrol. For these composts, Bacillus spp., oligotrophic and cellulolytic actinomycetes, and oligotrophic actinomycetes/oligotrophic bacteria and cellulolytic actinomycetes/cellulolytic bacteria ratios were indicated as microbial populations potentially involved in suppression.     

Potential for plant growth promotion in groundnut (Arachis hypogaea L.) cv. ALR-2 by co-inoculation of sulfur-oxidizing bacteria and Rhizobium

The use of Rhizobium inoculant for groundnut is a common practice in India. Also, co-inoculation of Rhizobium with other plant growth-promoting bacteria received considerable attention in legume growth promotion. Hence, in the present study we investigated effects of co-inoculating the sulfur (S)-oxidizing bacterial strains with Rhizobium, a strain that had no S-oxidizing potential in groundnut. Chemolithotrophic S-oxidizing bacterial isolates from different sources by enrichment isolation technique included three autotrophic (LCH, SWA5 and SWA4) and one heterotrophic (SGA6) strains. All the four isolates decreased the pH of the growth medium through oxidation of elemental S to sulfuric acid. Characterization revealed that these isolates tentatively placed into the genus Thiobacillus. Clay-based pellet formulation (2.5 x 10(7) cf ug(-1) pellet) of the Thiobacillus strains were developed and their efficiency to promote plant growth was tested in groundnut under pot culture and field conditions with S-deficit soil. Experiments in pot culture yielded promising results on groundnut increasing the plant biomass, nodule number and dry weight, and pod yield. Co-inoculation of Thiobacillus sp. strain LCH (applied at 60 kg ha(-1)) with Rhizobium under field condition recorded significantly higher nodule number, nodule dry weight and plant biomass 136.9 plant(-1), 740.0mg plant(-1) and 15.0 g plant(-1), respectively, on 80 days after sowing and enhanced the pod yield by 18%. Also inoculation of S-oxidizing bacteria increased the soil available S from 7.4 to 8.43 kg ha(-1). These results suggest that inoculation of S-oxidizing bacteria along with rhizobia results in synergistic interactions promoting the yield and oil content of groundnut, in S-deficit soils.