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ABSTRACT: High concentrations of indole are known to be toxic to cells due to perturbations in membrane potential. Here, we report for the first time a transcriptome analysis of a soil model bacterium, Pseudomonas putida KT2440, under indole treatment. We demonstrated that 47 genes are differentially expressed, including 11 genes involved in the tricarboxylic acid cycle (TCA cycle) and 12 genes involved in chaperone and protease functions (hslV, hslU, htpG, grpE, dnaK, ibpA, groEL, groES, clpB, lon-1, lon-2, and hflk). Mutant analysis supported the observation that protease genes including hslU are essential for the indole resistance of Pseudomonas strains. Subsequent biochemical analyses have shown that indole increases the NADH/NAD(+) ratio and decreases the adenosine triphosphate (ATP) concentration inside cells, due to membrane perturbation and higher expression of TCA cycle genes in the presence of indole. This energy reduction leads to a reduction in cell size and an enhancement of biofilm formation in P. putida. The observed upregulation in many chaperones and proteases led us to speculate that protein folding might be inhibited by indole treatment. Interestingly, our in vitro protein-refolding assay using malate dehydrogenase with purified GroEL/GroES demonstrated that indole interferes with protein folding. Taken together, our data provides new evidence that indole causes toxicity to Pseudomonas putida by inhibiting cellular energy production and protein folding. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
FEMS Microbiology Letters 03/2013; · 2.04 Impact Factor
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ABSTRACT: The effects of malachite green (MG) on the bacterial community in Antarctic soil were assessed. Culture-independent community analysis using 16S rRNA gene pyrosequencing showed that, in the presence of MG, the relative abundance of Pseudomonas dramatically increased from 2.2 % to 36.6 % (16.6-fold), and Pseudomonas became the predominant genus. The reduction in bacterial biodiversity was demonstrated by diversity indices and rarefaction curves. MG-degrading Pseudomonas sp. MGO was isolated from Antarctic soil. MG tolerance and decolorization activity were confirmed by growth, spectrophotometric, high-performance liquid chromatography, and thin-layer chromatography analyses in high MG concentrations. Our data showed that the decolorization process occurred via biodegradation, while biosorption also occurred after some time during the fed-batch decolorization process. Significant inductions in laccase, nicotinamide adenine dinucleotide-2,6 dichlorophenol indophenol reductase, and MG reductase activities suggested their involvement in the decolorization process. We also showed that the high tolerance of strain MGO to toxic MG might be mediated by upregulation of oxidative stress defense systems such as superoxide dismutase and protease. Collectively, these results demonstrated the response of the Antarctic soil bacterial community to MG and provided insight into the molecular mechanism of MG-tolerant Pseudomonas strains isolated from Antarctic soil.
Applied Microbiology and Biotechnology 01/2013; · 3.42 Impact Factor
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ABSTRACT: Pollution of Antarctic soils may be attributable to increased nutritional input and diesel contamination via anthropogenic activities. To investigate the effect of these environmental changes on the Antarctic terrestrial ecosystem, soil enzyme activities and microbial communities in 3 types of Antarctic soils were evaluated. The activities of alkaline phosphomonoesterase and dehydrogenase were dramatically increased, whereas the activities of β-glucosidase, urease, arylsulfatase, and fluorescein diacetate hydrolysis were negligible. Alkaline phosphomonoesterase and dehydrogenase activities in the 3 types of soils increased 3- to 10-fold in response to nutritional input, but did not increase in the presence of diesel contamination. Consistent with the enzymatic activity data, increased copy numbers of the phoA gene, encoding an alkaline phosphomonoesterase, and the 16S rRNA gene were verified using quantitative real-time polymerase chain reaction. Interestingly, dehydrogenase activity and 16S rRNA gene copy number increased slightly after 30 days, even under diesel contamination, probably because of adaptation of the bacterial population. Intact Antarctic soils showed a predominance of Actinobacteria phylum (mostly Pseudonorcarida species) and other phyla such as Proteobacteria, Chloroflexi, Planctomycetes, Firmicutes, and Verrucomicrobia were present in successively lower proportions. Nutrient addition might act as a selective pressure on the bacterial community, resulting in the prevalence of Actinobacteria phylum (mostly Arthrobacter species). Soils contaminated by diesel showed a predominance of Proteobacteria phylum (mostly Phyllobacterium species), and other phyla such as Actinobacteria, Bacteroidetes, Planctomycetes, and Gemmatimonadetes were present in successively lower proportions. Our data reveal that nutritional input has a dramatic impact on bacterial communities in Antarctic soils and that diesel contamination is likely toxic to enzymes in this population.
The Journal of Microbiology 12/2012; 50(6):916-24. · 1.10 Impact Factor
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ABSTRACT: The nosZ gene encodes nitrous oxide reductase, a key enzyme in nitrous oxide reduction occurring during complete denitrification. Many conventional approaches have used Proteobacteria-based primers to detect nosZ in environmental samples. However, these primers often fail to detect nosZ in non-Proteobacteria strains, including Firmicutes (gram-positive) and Bacteroidetes. In this study, newly designed nosZ primers successfully amplified this gene from 5 Geobacillus species (Firmicutes). The primers were used to construct nosZ clone libraries from DNA extracted from sludge and domestic animal feedlot soils, all with high organic carbon contents. After DNA sequencing, phylogenetic analysis identified many new nosZ sequences with high levels of homology to nosZ from Bacteroidetes, probably because of the high sequence similarity of nosZ from Firmicutes and Bacteroidetes, and a predominance of Bacteroidetes in feedlot environments. Three sets of new qPCR primers based on our clone library sequences were designed and tested for their specificities. Our data showed that only Bacteroidetes-related nosZ sequences were amplified, whereas conventional Proteobacteria-based primers amplified only Proteobacteria-related nosZ. Quantitative analysis of nosZ with the new qPCR primers recovered ~104 copies/100ng DNA. Thus, it appears that amplification with conventional primers is insufficient for developing an understanding of the diversity and abundance of nosZ genes in the environment.
Microbiology 11/2012; · 3.06 Impact Factor
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ABSTRACT: Staphylococcus sp. strain OJ82 was isolated from a Korean traditional fermented squid seafood, ojingeo-jeotgal. Staphylococcus sp. OJ82 could grow and show extracellular protease and β-galactosidase activities in the presence of extremely high saline (20%). Here, we report the genome sequence of Staphylococcus sp. OJ82.
Journal of bacteriology 11/2012; 194(22):6353-4. · 3.94 Impact Factor
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ABSTRACT: We present the genome sequence of Alishewanella aestuarii B11(T) (=KCTC 22051(T)=DSM 19476(T)). This species, isolated from tidal flat sediment, was reported to be a novel species. A. aestuarii is known to degrade pectin, an important component of plant cell wall. The presence of the genes related to pectin metabolism in this strain indicates its capability to utilize pectin.
Journal of bacteriology 10/2012; 194(19):5476. · 3.94 Impact Factor
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ABSTRACT: Alishewanella agri BL06(T) (= KCTC 22400(T) = JCM 15597(T)) was isolated from landfill soil in Pohang, South Korea. A. agri showed the ability to degrade pectin, a structural heteropolysaccharide present in the cell wall of plants. Here we report the genome sequence of Alishewanella agri BL06(T), the second sequenced strain in the genus Alishewanella.
Journal of bacteriology 09/2012; 194(18):5135-6. · 3.94 Impact Factor
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ABSTRACT: Flavodoxin (Fld) has been demonstrated to bind to ferredoxin- NADP(+) reductase A (FprA) in Pseudomonas putida. Two residues (Phe(256), Lys(259)) of FprA are likely to be important for interacting with Fld based on homology modeling. Sitedirected mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-Ala(259) and FprA-Asp(259) proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-Ala(259) and FprA-Asp(259) were examined by isothermal titration calorimetry. We demonstrated, for the first time, that Phe(256) and Lys(259) are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex.
BMB reports 08/2012; 45(8):476-81. · 1.72 Impact Factor
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ABSTRACT: Strain BH45(T) was isolated from forest soil of Mt. Bukhan in Jeongneung, Seoul, Korea. The Gram-staining-negative strain BH45(T) grows at 4-30°C (optimum of 25-30°C) and between pH 5-8 (optimum of pH 6-8). Its major cellular fatty acids are C(18:3) ω6c (6,9,12) and C(10:0). The G+C content of genomic DNA was 40.2 mol%. The major respiratory quinone system in strain BH45(T) is menaquinone-7. Phylogenetic analysis based on 16S rRNA gene sequences indicates that strain BH45(T) is closely related to the genus Pedobacter. Sequence similarities with P. terrae KCTC 12762(T), P. suwonensis KACC 11317(T), P. soli KACC 14939(T), P. alluvionis DSM 19624(T), P. roseus KCCM 42272(T), P. yonginense KCTC 22721(T) were 97.5, 97.1, 97.0, 97.0, 97.0, and 96.0%, respectively. DNA-DNA hybridization results distinguish strain BH45(T) from two Pedobacter species with high 16S rRNA gene sequence similarities. According to the phenotypic and molecular data, the strain BH45(T) clearly represents a novel species within the genus Pedobacter; thus, the name Pedobacter jeongneungensis sp. nov. is proposed for this strain. The type strain is BH45(T) (=KACC 15514(T) =JCM 17626(T)).
The Journal of Microbiology 08/2012; 50(4):660-4. · 1.10 Impact Factor
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ABSTRACT: Flavodoxin (Fld) is a bacterial electron-transfer protein that possesses flavin mononucleotide as a prosthetic group. In the genomes of the Pseudomonas species, the mioC gene is the sole gene, annotated Fld, but its function remains unclear. In this study, phenotype microarray analysis was performed using the wild-type and mioC mutant of pathogenic Pseudomonas aeruginosa PAO1. Our results showed that the mioC mutant is very resistant to oxidative stress. Different antibiotics and metals worked differently on the sensitivity of the mutant. Other pleiotropic effects of mutation in the mioC gene, such as biofilm formation, aggregation ability, motility and colony morphology, were observed under iron stress conditions. Most of the phenotypic and physiological changes could be recovered in the wild type by complementation. Mutation of the mioC gene also influenced the production of pigments. The mioC mutant and mioC over-expressed complementation cells, over-produced pyocyanin and pyoverdine, respectively. Various secreted chemicals were also changed in the mutant, which was confirmed by (1) H NMR analysis. Interestingly, physiological alterations of the mutant strain were restored by the cell-free supernatant of the wild type. The present study demonstrates that the mioC gene plays an important role in the physiology of P. aeruginosa and might be considered as a suitable drug target candidate in pathogenic P. aeruginosa.
FEMS Microbiology Letters 07/2012; 335(1):47-57. · 2.04 Impact Factor
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ABSTRACT: The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change.
The Journal of Microbiology 06/2012; 50(3):365-73. · 1.10 Impact Factor
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ABSTRACT: The oxyR gene appears to reside in an operon with the recG helicase gene in many bacteria, including pathogenic Pseudomonas aeruginosa and Pseudomonas putida. Analysis of P. putida transcriptomes shows that many OxyR-controlled genes are regulated by the ATP-dependent RecG helicase and that RecG alone modulates the expression of many genes. We found that purified RecG binds to the promoters of many OxyR-controlled genes and that expression of these genes was not induced under conditions of oxidative stress in recG mutants of P. aeruginosa, P. putida, and Escherichia coli. In vitro data revealed that promoters containing palindromic sequences are essential for RecG binding and that single-strand binding proteins and ATP are also needed for RecG to promote transcription, whereas a magnesium ion has the opposite effect. The OxyR tetramer preferentially binds to promoters after RecG has generated linear DNA in the presence of ATP; otherwise, the OxyR dimer has higher affinity. This study provides new insights into the mechanism of bacterial transcription by demonstrating that RecG might be required for the induction of the OxyR regulon by unwinding palindromic DNA for transcription. This work describes a novel bacterial transcriptional function by RecG helicase with OxyR and may provide new targets for controlling Pseudomonas species pathogen.
Journal of Biological Chemistry 05/2012; 287(29):24492-504. · 4.77 Impact Factor
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ABSTRACT: Alishewanella jeotgali MS1(T) (= KCTC 22429(T) = JCM 15561(T)) was isolated from a traditional Korean fermented seafood, gajami sikhae (jeotgal), and has been reported as a novel species. A. jeotgali was proven to have extracellular proteolytic activity, which may play an important role in the fermentation environment of food containing fish flesh. Here, we present the genome sequence of Alishewanella jeotgali MS1(T) as the first sequenced strain in the genus Alishewanella and its taxonomic relatives.
Journal of bacteriology 04/2012; 194(8):2097. · 3.94 Impact Factor
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ABSTRACT: A Gram-staining-negative, yellow-pigmented, strictly aerobic bacterium, designated strain SC17T, was isolated from sediment of a tidal flat of the Suncheon bay in South Korea. Cells were halotolerant, catalase- and oxidase-positive and non-motile rods. Growth of strain SC17T was observed at 5-40 °C (optimum, 25-30 °C), at pH 6.0-8.5 (optimum, pH 7.0) and in the presence of 1-8 % (w/v) NaCl (optimum, 1-2 %). The major cellular fatty acids were iso-C15:0 (26.0 %), summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c and/or iso-C15:0 2-OH; 11.7 %), iso-C17:0 3-OH (10.1 %), iso-C15:1 G (9.6 %) and anteiso-C15:0 (9.5 %). The polar lipid content consisted of phosphatidylethanolamine and unidentified amino lipids and lipids. The G+C content of the genomic DNA was 46.4 mol% and the respiratory quinone detected was only menaquinone 6 (MK-6). Phylogenetic inference based on 16S rRNA gene sequences showed that strain SC17T formed a distinct phyletic lineage within the family Flavobacteriaceae and was most closely related to members of the genera Gaetbulibacter and Tamlana with 95.0-95.8 % sequence similarity. On the basis of phenotypic and molecular features, strain SC17T represents a novel genus of the family Flavobacteriaceae, for which the name Aestuariibaculum suncheonense gen. nov., sp. nov. is proposed. The type strain is SC17T (=KACC 16186T = JCM 17789T). Emended descriptions of the genera Gaetbulibacter and Tamlana are also proposed.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 03/2012; · 2.11 Impact Factor
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ABSTRACT: The addition of non-ionic solutes such as sucrose and polyethylene glycol (PEG) to a culture of Escherichia coli O157:H7 stimulated formation of a biofilm on an abiotic surface. Possible factors involved in this increased biofilm formation were evaluated, i.e. oxidative stress, exopolysaccharide (EPS) production, membrane composition and lipopolysaccharide (LPS) production. A green fluorescent protein (GFP)-based reporter strain, anaerobic experiment and microarray data suggested that the increased biofilm formation was not due to oxidative stress. Quantification of the EPS revealed that cell-released EPS production appeared not to be related. Bacterial results of fatty acid methyl ester (FAME) analysis, along with microarray data, showed that sucrose and PEG could induce membrane rigidity via alterations in the fatty acid (FA) composition. Based on transcriptome analysis, PEG was observed to induce several membrane-related genes and membrane-associated LPS synthesis genes, confirmed by quantitative real-time RT-PCR analysis. Interestingly, biofilm cells showed higher expression than planktonic cells of ompC (encoding an outer membrane protein) and many LPS- and polysaccharide-related genes (glmS, dxs, msbB and kdsA genes) when subjected to PEG treatment. Greater LPS production could be observed under both PEG and sucrose-added biofilm conditions in E. coli O157:H7. Our data suggest that sucrose and PEG resulted in biofilm formation of E. coli O157:H7, not as a result of oxidative stress and EPS production, but via increases in membrane rigidity and LPS production.
Research in Microbiology 02/2012; 163(4):258-67. · 2.76 Impact Factor
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ABSTRACT: BACKGROUND: Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae poses serious challenges to clinicians because of its resistance to many classes of antibiotics. METHODS AND FINDINGS: The mechanism of synergistic activity of a combination of (-)-epigallocatechin-3-gallate (EGCG) and β-lactam antibiotics cefotaxime was studied on Extended-spectrum β-lactamase producing Escherichia coli (ESBL-EC), by visualizing the morphological alteration on the cell wall induced by the combination using atomic force microscopy (AFM). Cells at sub-MICs (sub-minimum inhibitory concentrations) of cefotaxime were initially filamentated but recovered to the normal shape later, whereas cells at sub-MICs of EGCG experienced temporal disturbance on the cell wall such as leakage and release of cellular debris and groove formation, but later recovered to the normal shape. In contrast, the combination of cefotaxime and EGCG at their respective sub-MICs induced permanent cellular damages as well as continuous elongation in cells and eventually killed them. Flow cytometry showed that intracellular oxidative stress levels in the cell treated with a combination of EGCG and cefotaxime at sub-MICs were higher than those in the cells treated with either cefotaxime or EGCG at sub-MICs. CONCLUSIONS: These results suggest that the synergistic effect of EGCG between EGCG and cefotaxime against ESBL-EC is related to cooperative activity of exogenous and endogenous reactive oxygen species (ROS) generated by EGCG and cefotaxime, respectively.
PLoS ONE 01/2012; 7(11):e48880. · 4.09 Impact Factor
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ABSTRACT: Alteromonas species are globally distributed copiotrophic bacteria in marine habitats. Among these, sea-tidal flats are distinctive: undergoing seasonal temperature and oxygen-tension changes, plus periodic exposure to petroleum hydrocarbons. Strain SN2 of the genus Alteromonas was isolated from hydrocarbon-contaminated sea-tidal flat sediment and has been shown to metabolize aromatic hydrocarbons there. Strain SN2's genomic features were analyzed bioinformatically and compared to those of Alteromonas macleodii ecotypes: AltDE and ATCC 27126. Strain SN2's genome differs from that of the other two strains in: size, average nucleotide identity value, tRNA genes, noncoding RNAs, dioxygenase gene content, signal transduction genes, and the degree to which genes collected during the Global Ocean Sampling project are represented. Patterns in genetic characteristics (e.g., GC content, GC skew, Karlin signature, CRISPR gene homology) indicate that strain SN2's genome architecture has been altered via horizontal gene transfer (HGT). Experiments proved that strain SN2 was far more cold tolerant, especially at 5°C, than the other two strains. Consistent with the HGT hypothesis, a total of 15 genomic islands in strain SN2 likely confer ecological fitness traits (especially membrane transport, aromatic hydrocarbon metabolism, and fatty acid biosynthesis) specific to the adaptation of strain SN2 to its seasonally cold sea-tidal flat habitat.
PLoS ONE 01/2012; 7(4):e35784. · 4.09 Impact Factor
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ABSTRACT: Acinetobacter oleivorans DR1 lacks an upper pathway for naphthalene degradation and cannot grow using naphthalene as sole carbon source; however, it is capable of growing under naphthalene-amended conditions in the presence of naphthalene-degrading Pseudomonas sp. AS1. ¹H-NMR spectroscopy, high-performance liquid chromatography and gene expression analyses showed that salicylate is a major secreted metabolic intermediate during naphthalene degradation by strain AS1 and that, in turn, it supports the growth of strain DR1. Interspecies biofilm formation, monitored using confocal laser scanning microscopy and microtiter assays, demonstrated that biofilm formation by strain AS1 increased dramatically in the presence of strain DR1 because of the exopolysaccharides generated by the latter. Furthermore, the metabolic commensal interaction of the two strains altered the initial attachment behavior of strain DR1 during biofilm formation. When this strain was cultivated alone under naphthalene-amended conditions, the cells immediately attached to the surface, probably due to the absence of usable substrates, whereas similar behavior was not observed in the mixed culture. This interspecies cell-cell interaction became more complex due to quenching of the quorum-sensing signal of strain DR1 by strain AS1. These complex metabolic and physiological interactions observed in mixed cultures suggest that interspecies interaction is more complicated than previously surmised.
Research in Microbiology 12/2011; 163(3):173-81. · 2.76 Impact Factor
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ABSTRACT: Zn(2+) uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficient conditions. ykgM and zinT (formerly yodA), regulated by Zur (zinc uptake regulator), have been reported as being highly induced during zinc shortage. This work reports that ykgM and zinT in enterohemorrhagic Escherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those in stationary-phase planktonic cells and a mutation of either ykgM or zinT genes led to the inhibition of curli biosynthesis. Inductively coupled plasma mass spectroscopy showed that the ykgM and zinT mutants contained lower concentrations of Zn(2+) than the wild type. Both mutants were less attached to both the glass surface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR data indicated that the expression of csgA, which encodes the major curli subunit, was inhibited in both mutants with a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficient condition were covered with a lower amount of curli compared to the wild type and often became filamentous. Zn(2+) supplementation restored curli production and prevented filamentation in the mutants. Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zinc concentration in EHEC and intracellular zinc deficiency inhibits curli production.
International journal of food microbiology 09/2011; 149(2):159-70. · 3.01 Impact Factor
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ABSTRACT: The comparative genomics of Acinetobacter oleivorans DR1 assayed with A. baylyi ADP1, A. calcoaceticus PHEA-2, and A. baumannii ATCC 17978 revealed that the incorporation of phage-related genomic regions and the absence of transposable elements have contributed to the large size (4.15 Mb) of the DR1 genome. A horizontally transferred genomic region and a higher proportion of transcriptional regulator- and signal peptide-coding genes were identified as characteristics of the DR1 genome. Incomplete glucose metabolism, metabolic pathways of aromatic compounds, biofilm formation, antibiotics and metal resistance, and natural competence genes were conserved in four compared genomes. Interestingly, only strain DR1 possesses gentisate 1,2-dioxygenase (nagI) and grows on gentisate, whereas other species cannot. Expression of the nagI gene was upregulated during gentisate utilization, and four downstream open reading frames (ORFs) were cotranscribed, supporting the notion that gentisate metabolism is a unique characteristic of strain DR1. The genomic analysis of strain DR1 provides additional insights into the function, ecology, and evolution of Acinetobacter species.
Applied and environmental microbiology 08/2011; 77(20):7418-24. · 3.69 Impact Factor
