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Gordonia sihwensis sp. nov., a novel nitrate-reducing bacterium isolated from a wastewater-treatment bioreactor.

A nitrate-reducing bacterium, strain SPR2(T), was isolated from a sulphur-oxidizing, autotrophic denitrification reactor used for advanced treatment of wastewater from the lake of Sihwa, Korea. The strain was aerobic but could grow under anaerobic conditions. It was Gram-positive, exhibited rough white colonies on complex nutrient agar, produced elementary branching hyphae that fragmented into rod/coccus-like elements and showed chemotaxonomic markers that were consistent with classification in the genus Gordonia, i.e. meso-diaminopimelic acid, arabinose and galactose in whole-cell hydrolysates, N-glycolylmuramic acid in the peptidoglycan wall, unbranched saturated and monounsaturated fatty acids plus tuberculostearic acid (TBSA), mycolic acids that comprised 48-56 carbon atoms and MK-9(H(2)) as the predominant menaquinone. The 16S rDNA sequence of strain SPR2(T) showed highest similarity to Gordonia amicalis DSM 44461(T) and Gordonia hydrophobica DSM 44015(T), with values of 98.2 and 97.9 %, respectively. These values were far below 99.5 % (usually found at the intraspecies level) and they were in the range that separates species at the intrageneric level. The separate phylogenetic position of SPR2(T) was supported by differences in fatty acid and mycolic acid compositions and in carbon utilization tests that distinguished strain SPR2(T) from all known Gordonia species. Combined genotypic and phenotypic data show that strain SPR2(T) merits recognition as a novel species within the genus Gordonia, for which the name Gordonia sihwensis sp. nov. is proposed; the type strain is SPR2(T) (=DSM 44576(T)=NRRL B-24155(T)).

Bioreactors↗

Novel acetone metabolism in a propane-utilizing bacterium, Gordonia sp. strain TY-5.

In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane --> 2-propanol --> acetone --> methyl acetate --> acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.

Acetone↗

Characterization of the 101-kilobase-pair megaplasmid pKB1, isolated from the rubber-degrading bacterium Gordonia westfalica Kb1.

The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1.

Biodegradation, Environmental↗

Desulfurization of dibenzothiophene, benzothiophene, and other thiophene analogs by a newly isolated bacterium, Gordonia alkanivorans strain 1B.

A novel bacterium, Gordonia alkanivorans strain 1B, was isolated from hydrocarbon-contaminated soil. Assessment of the biodegradation of distinct organic sulfur-compounds, such as dibenzothiophene (DBT), benzothiophene (BT), DBT sulfone, and alkylated tiophenic compounds, as the sole source of sulfur was investigated. G. alkanivorans strain 1B was able to remove selectively the sulfur from DBT while keeping intact the remaining carbon-carbon structure. Orthophenyl phenol (2-hydroxybiphenyl) was the only detected metabolic product. The bacterial desulfurization activity was repressed by sulfate. G. alkanivorans strain 1B consumed 310 microM DBT after 120 h of cultivation, corresponding to a specific desulfurization rate of 1.03 micromol/(g of dry cells x h). When an equimolar mixture of DBT/BT was used as a source of sulfur in the growth medium, G. alkanivorans strain 1B assimilated both compounds in a sequential manner, with BT as the preferred source of sulfur. Only when BT concentration was decreased to a very low level was DBT utilized as the source of sulfur for bacterial growth. The specific desulfurization overall rates of BT and DBT obtained were 0.954 and 0.813 micromol/(g of dry cells x h), respectively. The newly isolated G. alkanivorans strain 1B has good potential for application in the biodesulfurization of fossil fuels.

Biodegradation, Environmental↗

Modifications of the follicle-associated epithelium by short-term exposure to a non-intestinal bacterium.

This study was undertaken in order to study the effects of short-term exposure of the follicle-associated epithelium (FAE) of rabbit Peyer's patches to a non-intestinal, Gram-positive bacterium. Isolated ileal loops, each containing one Peyer's patch (PP), were stimulated for short periods of time (30 and 60 min) with Streptococcus pneumoniae R36a, a micro-organism normally not present in the intestinal area. Samples from antigen-stimulated and control Peyer's patches were analysed by light (LM), transmission electron (TEM), and scanning electron microscopy (SEM). Stimulation with living pneumococci induced dramatic changes in FAE architecture and morphology. A massive passage of cells from lymphoid tissue to the FAE was rapidly detectable, accompanied by alterations of the FAE surface, with a marked increase of M-cell area. Furthermore, TEM analysis revealed that M cells were able to internalize living pneumococci. S. pneumoniae R36a is a valid experimental model for the further study of the unique antigen sampling function which characterizes the highly specialized FAE in Peyer's patches.

Animals↗

Effects of resin composite components on glucosyltransferase of cariogenic bacterium.

The aim of our study was to investigate the effect of resin composite components on glucan synthesis by glucosyltransferase (GTase) derived from a cariogenic bacterium, Streptococcus sobrinus B13. The eluates from cured composites stored in 0.05 mol/L potassium phosphate buffer (KPB) (pH 6.8) for 2 weeks at 37 degrees C stimulated the formation of water-insoluble glucan, whereas those from amalgam inhibited it. This finding suggests that the eluates from the resin composites enhance GTase activity and contribute to plaque formation. In the individual resin components, a diglycidyl methacrylate and triethylene glycol dimethacrylate significantly enhanced GTase activity. However, most polymerization inhibitors and accelerators were potent inhibitors of enzyme activity. Thus, GTase stimulation by eluates from resin composites is hypothesized to be heavily dependent on the effect of the eluated resin monomers, even though the other eluting components had inhibitory effects on GTase.

Composite Resins↗

Effect of carbon and nitrogen sources on growth dynamics and exopolysaccharide production for the hyperthermophilic archaeon Thermococcus litoralis and bacterium Thermotoga maritima.

Batch and continuous cultures were used to compare specific physiological features of the hyperthermophilic archaeon, Thermococcus litoralis (T(opt) of 85 degrees to 88 degrees C), to another fermentative hyperthermophile that reduces S degrees facultatively, that is, the bacterium Thermotoga maritima (T(opt) of 80 degrees to 85 degrees C). Under nutritionally optimal conditions, these two hyperthermophiles had similar growth yields on maltose and similar cell formula weights based on elemental analysis: CH(1.7)O(0. 7)N(0.2)S(0.006) for T. litoralis and CH(1.6)O(0.6)N(0.2)S(0.005) for T. maritima. However, they differed with respect to nitrogen source, fermentation product patterns, and propensity to form exopolysaccharides (EPS). T. litoralis could be cultured in the absence or presence of maltose on an amino acid-containing defined medium in which amino acids served as the sole nitrogen source. T. maritima, on the other hand, did not utilize amino acids as carbon, energy, or nitrogen sources, and could be grown in a similar defined medium only when supplemented with maltose and ammonium chloride. Not only was T. litoralis unable to utilize NH(4)Cl as a nitrogen source, its growth was inhibited at certain levels. At 1 g/L ( approximately 20 mM) NH(4)Cl, the maximum growth yield (Y(x/s(max))) for T. litoralis was reduced to 13 g cells dry weight (CDW)/mol glucose from 40 g CDW/mol glucose in media lacking NH(4)Cl. Alanine production increased with increasing NH(4)Cl concentrations and was most pronounced if growth on NH(4)Cl was carried out in an 80% H(2) atmosphere. In T. maritima cultures, which would not grow in an 80% H(2) atmosphere, alanine and EPS were produced at much lower levels, which did not change with NH(4)Cl concentration. EPS production rose sharply at high dilution rates for both organisms, such that maltose utilization plots were biphasic. Wall growth effects were also noted, because cultures failed to wash out at dilution rates significantly above maximum growth rates determined from batch growth experiments. This study illustrates the importance of effective cultivation methods for addressing physiological issues related to the growth of hyperthermophilic heterotrophs.

Amino Acids↗

Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach.

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.

Acetylglucosamine↗

The proteome of the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products.

An existing proteome map of the bacterium Mycoplasma pneumoniae comprising proteins from 224 genes was extended to 305 genes. This corresponds to about 44% of the 688 proposed genome sequence derived open reading frames (ORFs). The newly assigned gene products were enriched, separated by one-dimensional or two-dimensional (2-D) gel electrophoresis and identified by mass spectrometry. The enrichment procedures included differential centrifugation, anion and cation exchange chromatography, affinity chromatography with heparin as a ligand and isolation of biotinylated proteins by binding to immobilized streptavidin. A comparative analysis of the identified proteins from 305 genes with the as yet unverified 383 ORFs concerning isoelectric point, molecular weight and number of transmembrane segments revealed that proteins with more than three predicted transmembrane segments and an isoelectric point above 10.5 are most likely not to be separated by 2-D gel electrophoresis. The mutual benefits of genomics and proteomics were shown by the identification of a todate unannotated 128 amino acid long protein.

Amino Acid Sequence↗

Testing the photosynthetic bacterium Rhodobacter sphaeroides as heavy metal removal tool.

We present some preliminary results relevant to the ability of the purple non-sulphur bacterium Rhodobacter sphaeroides strain R26.1 to sequester heavy metals from contaminated growth media. The microorganism was chosen because of its significant tolerance to relatively high concentrations of the investigated ions Ni2+, Co2+, CrO4(2-), and MoO4(2-). In this paper the optimized conditions for the bacterial growth and the sample preparation used to infer the ability of the microorganism to cope with metal pollutants are presented. Elemental analysis has been performed by inductively coupled plasma atomic emission spectrometry previous mineralization of samples by a microwave system.

Environmental Pollutants↗

Effects of a strong static magnetic field on bacterium Shewanella oneidensis: an assessment by using whole genome microarray.

The effect of a strong static 14.1 T magnetic field on log phase cells of bacterial strain Shewanella oneidensis MR-1 was evaluated by using whole genome microarray of this bacterium. Although differences were not observed between the treatment and control by measuring the optical density (OD), colony forming unit (CFU), as well as post-exposure growth of cells, transcriptional expression levels of 65 genes were altered according to our microarray data. Among these genes, 21 were upregulated while other 44 were downregulated, compared with control.

Bacterial Proteins↗

Inhibition of light emission from the bioluminescent bacterium Vibrio fischeri after exposure to triclosan and related hygiene care products.

The affect of the anti-microbial agent triclosan (alternative names Microban and Irgasan DP300) on the light emission by the bioluminescent bacterium Vibrio fischeri was determined. Triclosan at concentrations greater than 0.2% (w/v) caused cell lysis and immediate (< 5 s) loss of light emission. Exposure to triclosan at lower concentrations caused a decrease in light output over time. The rate of the decrease in light output followed a cuboid relationship, of which the initial rate (first 60 s) of light loss was proportional to the concentration of triclosan. The effect on light output by two commercially available hygiene products containing triclosan also caused a similar response in light loss.

Anti-Infective Agents, Local↗

Removal of soluble selenium by a selenate-reducing bacterium Bacillus sp. SF-1.

In order to develop a biological process for removal of selenium from industrial wastewater, Bacillus sp. strain SF-1 was isolated from selenium-contaminated sediment. The bacterium reduces selenate to selenite and subsequently to nontoxic insoluble elemental selenium using lactate as an electron donor and selenate as an electron acceptor in an anaerobic condition. Elemental selenium transformed from soluble selenium was deposited both inside and outside of the cells. Since the selenate reduction rate of the strain SF-1 was higher than the selenite reduction rate, selenite was transiently accumulated. In an experiment of the repeated soluble selenium reduction by strain SF-1, 0.5 mM of selenate was sequentially treatable with a cycle of one day. Thus, our sequential system for removal of soluble selenium is very useful.

Bacillus↗

Inhibition of bacterial attachment by pulsed Nd:YAG laser irradiations: an in vitro study using marine biofilm-forming bacterium Pseudoalteromonas carrageenovora.

The effect of low mean power laser irradiations with short pulse duration from an Nd:YAG (neodymium-doped yttrium aluminium garnet) laser on a marine biofilm-forming bacterium, Pseudoalteromonas carrageenovora, was investigated in the laboratory. Laser-irradiated bacteria were tested for their ability to attach on nontoxic titanium nitride (TiN) coupons with nonirradiated bacteria as the reference. Two durations of irradiation were tested, 10 and 15 min. Bacterial attachment was monitored after 20 min, 40 min, and 1 h of irradiation. The average laser fluence used for this study was 0.1 J/cm(2). The area of attachment of the irradiated bacteria was significantly less than the reference for both durations of irradiation. The growth of irradiated bacteria showed a longer lag phase than the nonirradiated sample, mainly due to mortality in the former. The bacterial mortality observed was 23.4 +/- 0.71 and 48.6 +/- 6.5% for 10- and 15-min irradiations, respectively. Thus, the results show that low-power pulsed laser irradiations resulted in a significant bacterial mortality and a reduced bacterial attachment on nontoxic hard surfaces.

Biofilms↗

Laboratory-scale continuous reactor for soluble selenium removal using selenate-reducing bacterium, Bacillus sp. SF-1.

A model continuous flow bioreactor (volume 0.5 L) was constructed for removing toxic soluble selenium (selenate/selenite) of high concentrations using a selenate-reducing bacterium, Bacillus sp. SF-1, which transforms selenate into elemental selenium via selenite for anaerobic respiration. Model wastewater contained 41.8 mg-Se/L selenate and excess lactate as the carbon and energy source; the bioreactor was operated as an anoxic, completely mixed chemostat with cell retention time between 2.2-95.2 h. At short cell retention times selenate was removed by the bioreactor, but accumulation of selenite was observed. At long cell retention times soluble selenium, both selenate and selenite, was successfully reduced into nontoxic elemental selenium. A simple mathematical model is proposed to evaluate Se reduction ability of strain SF-1. First-order kinetic constants for selenate and selenite reduction were estimated to be 2.9 x 10(-11) L/cells/h and 5.5 x 10(-13) L/cells/h, respectively. The yield of the bacterial cells by selenate reduction was estimated to be 2.2 x 10(9) cells/mg-Se.

Bacillus↗

Laser impact assessment in a biofilm-forming bacterium Pseudoalteromonas carrageenovora using a flow cytometric system.

Impact by pulsed laser irradiations from an Nd:YAG laser on the marine biofilm-forming bacterium Pseudoalteromonas carrageenovora has been studied using a flow cytometric system. The biofilm-forming bacteria in the planktonic state have been irradiated while flowing, and the mortality and bacterial attachment have been determined by exposing TiN coupons in the system. Coupons suspended in the non-irradiated bacterial flow were treated as the control. The fluence used in the study was 0.1 J/cm(2). Three flow rates (14, 28, and 42 cm/min) and two exposure durations (15 and 30 min) were tested. The results showed the increase in bacterial mortality with the decrease in flow rate. The maximum mortality of 27.5% was observed when the flow rate was 14 cm/min. The bacterial attachment increased with the increase in flow rate and exposure duration. The area of bacterial attachment on the experimental coupons exposed to the irradiated sample was significantly lesser than that for the nonirradiated sample. The results thus show in a flowing system, low power pulsed laser irradiations could reduce the bacterial attachment even though it did not cause significant mortality.

Bacterial Adhesion↗

Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium.

Here we report the successful implementation of the Plackett-Burman multifactorial design to screen the limiting components for growth and subsequent use of the response surface methodology (RSM) to design a medium that supported exponential growth of the aggregated morphology of the shipworm bacterium, Teredinobacter turnirae. The results obtained with the help of Plackett-Burman design indicated limitations of three components in the growth medium, MnCl2.4H2O, Na2CO3, and K2HPO4. The concentrations of these three components were further optimized using RSM. By increasing the concentrations of the above-mentioned components by 4-fold, 12-fold, and 12-fold, respectively, it became possible to achieve exponential growth of the culture.

Algorithms↗