Comparative analysis of 2,4,6-trinitrotoluene (TNT)-induced cellular responses and proteomes in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 in two types of media

TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.     

Biological removal of explosive 2,4,6-trinitrotoluene byStenotrophomonas sp. OK-5 in bench-scale bioreactors

The biological removal of 2,4,6-trinitrotoluene (TNT) was studied in a bench-scale bioreactor using a bacterial culture of strain OK-5 originally isolated from soil samples contaminated with TNT. The TNT was completely removed within 4 days of incubation in a 2.5 L benchscale bioreactor containing a newly developed medium. The TNT was catabolized in the presence of different supplemented carbons. Only minimal growth was observed in the killed controls and cultures that only received TNT during the incubation period. This catabolism was affected by the concentration ratio of the substrate to the biomass. The addition of various nitrogen sources produced a delayed effect for the TNT degradation. Tween 80 enhanced the degradation of TNT under these conditions. Two metabolic intermediates were detected and identified as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene based on HPLC and GC-MS analyses, respectively. Strain OK-5 was characterized using the BIOLOG system and fatty acid profile produced by a microbial identification system equipped with a Hewlett packard HP 5890 II gas chromatograph. As such, the bacterium was identified as aStenotrophomonas species and designated asStenotrophomonas sp. OK-5.     

Enhanced Degradation of TNT by Genome-Shuffled Stenotrophomonas maltophilia OK-5

In this study, the enhanced degradation of TNT using cultures of genome-shuffled Stenotrophomonas maltophilia OK-5 mt-3 has been examined and the proteome of shuffled strain was compared to the wild-type OK-5 strain. Genome shuffling of S. maltophilia OK-5 was used to achieve a rapid enhancement of TNT degradation. The initial mutant population was generated by NTG treatment and UV irradiation. The wild-type OK-5 strain was able to degrade 0.2 mM TNT within 6 days, yet barely tolerated 0.5 mM TNT while the shuffled OK-5 mt-3 was capable of completely degrading 0.5 mM TNT within 8 days, and 1.2 mM within 24 days. The proteomic analysis of the shuffled OK-5 mt-3 demonstrated the changes in the expression levels of certain proteins compared to wild-type OK-5. These results provide clues for understanding TNT tolerance and improved TNT degradation by shuffled S. maltophilia OK-5 mt-3 and have possible applications in the processing of industrial waste containing relatively high TNT concentrations.     

Characterization of<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

The cellular responses of Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have been extensively analyzed in this study. The stress shock proteins, which might contribute to enhancing the cellular resistance to the cytotoxic effect of RDX, were induced at different concentrations of RDX used as a substrate for cell culture of Pseudomonas sp. HK-6. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using the anti-DnaK and anti-GroEL monoclonal antibodies. The stress shock proteins induced by RDX were found to increase in proportion to the RDX concentration used for this work. Analysis of membrane fatty acids of strain HK-6 following exposure to RDX showed that the amounts of dominant lipids 16:1 7c/15:0 iso 2OH, 16:0 and 18:1 7c/9t/12t decreased substantially or were not detected in the cells exposed to RDX, while amounts of lipids 10:0 iso, 14:1 5c/5t and 16:10 methyl increased dramatically. Scanning electron microcopy analyses revealed the presence of perforations and irregular rod shapes with wrinkled surfaces for cells treated with 0.135 mM RDX for 12 h, suggesting that RDX has a substantial cytotoxic impact on cells of strain HK-6.     

Induction of Stress Shock Proteins DnaK and GroEL by Phenoxyherbicide 2,4-D in Burkholderia sp. YK-2 Isolated from Rice Field

The purpose of this work was to investigate the induction of stress shock proteins in Burkholderia sp. YK-2 in response to the phenoxyherbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The stress shock proteins, which contribute to the resistance of the cytotoxic effect of 2,4-D, were induced at different 2,4-D concentrations in exponentially growing cultures of Burkholderia sp. YK-2. This response involved the induction of a 43-kDa DnaK and 41-kDa GroEL proteins, characterized by SDS-PAGE and Western blot by use of the anti-DnaK and anti-GroEL monoclonal antibodies. The total stress shock proteins were analyzed by 2-D PAGE. Survival of Burkholderia sp. YK-2 with time in the presence of different concentrations of 2,4-D was monitored, and viable counts paralleled the induction of the stress shock proteins in this strain. Received: 1 November 1999 / Accepted: 24 November 1999     

Membrane fatty acids adaptive profile in the simultaneous presence of arsenic and toluene in <Emphasis Type="Italic">Bacillus</Emphasis> sp. ORAs2 and <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. ORAs5 strains

Bacillus sp. ORAs2 and Pseudomonas sp. ORAs5, two arsenic-resistant bacterial strains previously isolated from sediments of the Orbetello Lagoon, Italy, were tested for their adaptation to mixed contaminants on the level of membrane fatty acid composition. The two bacterial strains were characterized by high levels of arsenic resistance, and Pseudomonas sp. ORAs5 was also shown to be solvent-tolerant. The bacterial strains were exposed to mixtures of two toxic compounds: arsenic at fixed concentrations and toluene in variable amounts or, alternatively, toluene at constant values along with arsenic added at variable concentrations. Both strains react to the contaminants by changing the composition of their membrane fatty acids. Bacillus sp. strain ORAs2 showed a correlation between growth rate decreases and fatty acids degree of saturation increases in both cases, although pointedly in the presence of 1, 2, and 3 mM of toluene and different additions of arsenic, counteracting membranes fluidity induced by toxic compounds. In Pseudomonas sp. ORAs5, adaptive changes in membrane composition was observed both in terms of increases in the degree of saturation and in the trans/cis ratio of unsaturated fatty acids in the presence of varying toluene and constant arsenic concentrations, whereas only minor changes occurred with increasing arsenic and constant toluene concentrations. Thus, on the level of membrane composition, Bacillus sp. ORAs2 showed a higher potential for adaptation to the presence of mixed pollutants, suggesting its probable suitability for bioremediation purposes.     

Physiological and Cellular Responses of the 2,4-D Degrading Bacterium, Burkholderia cepacia YK-2, to the Phenoxyherbicides 2,4-D and 2,4,5-T

Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp. YK-2 in response to sublethal concentrations of 2,4-D stress [Cho et al. (2000) Curr Microbiol 41:33–38]. In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T. Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T. SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide. These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells. Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T. Strain YK-2 could degrade 2.25 mM 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested. BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B. cepacia YK-2. Received: 7 February 2002 / Accepted: 7 March 2002     

Changes in protein composition ofSaccharomyces brewing strains in response to heat shock and ethanol stress

Summary Heat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.     

Cellular and proteomic responses of <Emphasis Type="Italic">Escherichia coli</Emphasis> JK-17 exposed to the <Emphasis Type="Italic">Rosa hybrida</Emphasis> flower extract

The purpose of this work was to characterize the cellular and proteomic responses of Escherichia coli JK-17 exposed to the rose flower extract (Rosa hybrida). The bacterial isolate was enriched and isolated from contaminated food. 16S rRNA sequence analyses revealed that the strain was 99% similar to the E. coli species cluster; therefore, this strain was designated E. coli JK-17. The rose flower extract showed a dose-dependent antibacterial effect on E. coli JK-17. Treatment of E. coli JK-17 with 50 and 100 mg/mL of the rose flower extract completely inhibited growth within 12 and 6 h of incubation. The stress shock proteins (SSPs) were induced with different concentrations of rose flower extract. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using anti-DnaK and anti-GroEL monoclonal antibodies. The levels of SSPs induced by the rose flower extract increased when the exposure time to the rose flower extract was increased. SDS-PAGE with silver staining revealed that the amount of lipopolysaccharide (LPS) in E. coli JK-17 increased or decreased with different concentrations and exposure times of the rose flower extract. To identify proteins induced by the rose flower extract, 2-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of E. coli JK-17 cultures. In the pH range of 4 ∼ 7, more than 250 spots were detected on the silver stained gels. Notably, 15 protein spots were increased or decreased after treatment with the rose flower extract. Twelve up-regulated proteins were identified as chaperones (DnaK and GroEL) and porin proteins (PhoE, RfaI, RfaG, MdoH, and WzzE) by MALDITOF mass spectrometry, and three down-regulated proteins were identified, including proteins involved in energy and DNA metabolism (SdhA and GyrB), and amino acid biosynthesis (GltK). Using scanning electron microscopic analysis, some cells were shown to adopt irregular rod shapes and wrinkled surfaces after treatment with the rose flower extract. These results provide clues for better understanding the mechanism of rose flower extract-induced stress and cytotoxicity in E. coli JK-17.     

Nitrogen effects on proteins, chlorophylls and fatty acids during the growth of Arthrospira platensis

Spirulina platensis (=Arthrospira platensis) is a tunisian strain which has been isolated for the first time in Oued Essed (Sousse, Sidi Bou Ali). Biomass evolution, proteins, chlorophylls and fatty acids composition of this alga were monitored by varying nitrogen concentrations in the culture medium. Nitrogen stress was provoked by adding sodium nitrate (NaNO3) in the culture medium with concentrations varying from 0 to 5 g/l. Results obtained showed that nitrogen depletion increased total proteins and total chlorophylls. The addition of NaNO3 (5g/l) led to an increase of total fatty acids amounts and modify fatty acids composition. Optimal quantities of palmitic, gamma -linolenic and oleic acids were obtained with NaNO3 free-cultures. Thus, the tunisian strain has valuable biological substances, worthy to determine the optimal conditions for its propagation.     

Physiological and cellular responses of the 2,4-D degrading bacterium,Burkholderia cepacia YK-2, to the phenoxyherbicides 2,4-D and 2,4,5-T

Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp. YK-2 in response to sublethal concentrations of 2,4-D stress [Cho et al. (2000) Curr Microbiol 41:33-38]. In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T. Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T. SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide. These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells. Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T. Strain YK-2 could degrade 2.25 m M 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested. BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B. cepacia YK-2.     

FATTY ACID AND LIPID COMPOSITION OF THE ACIDOPHILIC GREEN ALGA CHLAMYDOMONAS SP.1

The lipid and fatty acid compositions of Chlamydomonas sp. isolated from a volcanic acidic lake and C. reinhardtii were compared, and the effects of pH of the medium on lipid and fatty acid components of Chlamydomonas sp. were studied. The fatty acids in polar lipids from Chlamydomonas sp. were more saturated than those of C. reinhardtii. The relative percentage of triacylglycerol to the total lipid content in Chlamydomonas sp. grown in medium at pH 1 was higher than that in other cells grown at higher pH. A probable explanation might be that Chlamydomonas sp. has two low pH adaptation mechanisms. One mechanism is the saturation of fatty acids in membrane lipids to decrease membrane lipid fluidity, and the other is the accumulation of triacylglycerol, as a storage lipid, to prevent the osmotic imbalance caused by high concentrations of H₂SO₄.     

Cellular Responses and Proteomic Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Exposed to Green Tea Polyphenols

The purpose of this study was to characterize the cellular response and proteomic analysis of Escherichia coli exposed to tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L). TPP showed a dose-dependent bactericidal effect on E. coli. Analysis of cell-membrane fatty acids of E. coli cultures treated with TPP identified unique changes in saturated and unsaturated fatty acids, whereas scanning electron microscopic analysis demonstrated the presence of perforations and irregular rod forms with wrinkled surfaces in cells treated with TPP. Two-dimensional polyacrylamide gel electrophoresis of soluble protein fractions from E. coli cultures exposed to TPP showed 17 protein spots increased or decreased by TPP. Nine upregulated proteins were identified (including GroEL and proteins involved in cellular defense, such as GyrA, RpoS, SodC, and EmrK), whereas the expression of eight proteins was downregulated by exposure to TPP (including proteins involved in carbon and energy metabolism, such as Eno, SdhA, and UgpQ, as well as those involved in amino-acid biosynthesis, such as GltK and TyrB). These results provide clues for understanding the mechanism of TPP-induced stress and cytotoxicity on E. coli.     

Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA–DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment. Received: April 6, 2000 / Accepted: May 8, 2000     

<Emphasis Type="Italic">Stenotrophomonas panacihumi</Emphasis> sp. nov., isolated from soil of a ginseng field

The study isolated a Gram-negative, rod-shaped, non-motile bacterium from the soil of a ginseng field in Daejeon, South Korea and characterized it to determine its taxonomic position. Phylogenetic analysis, based on the 16S rRNA gene sequence, revealed that strain MK06^T belongs to the family Xanthomonadacea, and showed the highest degree of sequence similarity to Stenotrophomonas rhizophila e-p10^T (98.6%), Xanthomonas campestris LMG 568T (98.0%), Stenotrophomonas maltophilia ATCC 1d3637^T (97.3%), and Stenotrophomonas humi R-32729^T (96.9%). Chemotaxonomic data revealed that strain MK06^T possesses ubiquinone Q-8 as the predominant respiratory lipoquinone, which is common in the genus Stenotrophomonas, and that the predominant fatty acids were 15:0 iso (41.1%), 15:0 anteiso (12.6%), and 17:1 iso ω9c (8.6%). The results of physiological and biochemical tests clearly demonstrated that strain MK06^T represents a distinct species and supported its affiliation with the genus Stenotrophomonas. Based on these data, MK06^T (KCTC, 22893^T; JCM, 16536^T; KEMB, 9004-002^T) should be classified as the type strain for a novel species, for which we propose the name Stenotrophomonas panacihumi sp. nov.     

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.     

Fatty Acid Patterns in <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. as a Marker for Nutritional Regimes and Temperature under Extremely Acidic Conditions

Fatty acid profiles were used to characterize nutritional pathways in Chlamydomonas sp. isolated from an acidic mining lake (pH 2.7). Surprisingly, profiles of Chlamydomonas sp. grown in the lab under photoautotrophic, mixotrophic, and heterotrophic conditions at in situ deep strata lake water temperatures (8°C) were very similar, polyunsaturated fatty acids including -linolenic acid (18:33) and 16:43 along with palmitic acid (16:0) being most abundant. Therefore, heterotrophic growth of Chlamydomonas sp. at low temperatures can result in high concentrations of polyunsaturated fatty acids, as previously only described for some psychrophilic bacteria. By contrast, the cultivation of isolated Chlamydomonas sp. at 20°C, reflecting surface water temperatures, provided fatty acid patterns characteristic of the nutrition strategy applied: the concentration of polyunsaturated fatty acids decreased when the growth pathway changed from photoautotrophic via mixotrophic to heterotrophic. Total fatty acid concentration also diminished in this order. Principal component analysis confirmed the significance of FA profiling to mirror nutritional pathways. Lake-water analysis revealed low concentrations of dissolved organic carbon, mainly consisting of polymeric fulvic acids that are unable to support heterotrophic growth of Chlamydomonas sp. Polymeric fulvic acids present in the deeper strata of the lake turned out to be formed in situ on the basis of organic monomers including reduced sulfur-containing ones, as revealed by thermochemolysis and pyrolysis. Growth of Chlamydomonas sp. in the deep chlorophyll maximum is therefore assumed to mainly result from photosynthesis, despite very low photon densities. Phytol-including metabolites proved to be significant biomarkers to indicate the nutritional pathway of Chlamydomonas sp. , -Dicarboxylic acids—light-induced degradation products of unsaturated fatty acids—appeared to be good indicators of photooxidative alterations to the algal species under study.     

Isolation and characterization of a marine algicidal bacterium against the harmful raphidophyceae <Emphasis Type="Italic">Chattonella marina</Emphasis>

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of ULjin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 μl/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.