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ABSTRACT: Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa3- and cbb3-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb3-type oxidase is the predominant system for respiration of oxygen (O2), especially when O2 is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb3-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa3-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb3- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa3-type oxidase and switch of the regulatory system for respiration.The ISME Journal advance online publication, 11 April 2013; doi:10.1038/ismej.2013.62.
The ISME Journal 04/2013; · 7.38 Impact Factor
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ABSTRACT: Shewanella oneidensis is a highly motile organism by virtue of a polar, glycosylated flagellum composed of flagellins FlaA and FlaB. In this study, the functional flagellin FlaB was isolated and analyzed with nanoLC-MS and -MS/MS. In combination with the mutational analysis, we proposed that the FlaB flagellin protein from S. oneidensis is modified at five serine residues with a series of novel O-linked post-translational modifications (PTMs) that differ from each other by 14 Da. These PTMs are composed in part of a 274 Da sugar residue that bears a resemblance to the nonulosonic acids. The remainder appears to be composed of a second residue whose mass varies by 14 Da depending on the PTM. Further investigation revealed that synthesis of the glycans initiates with PseB and PseC, the first two enzymes of the Pse pathway. In addition, a number of lysine residues are found to be methylated by SO4160, an analogue of the lysine methyltransferase of Salmonella enterica serovar Typhimurium.
Journal of bacteriology 03/2013; · 3.94 Impact Factor
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ABSTRACT: Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (cyclic AMP receptor protein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp-independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.
Environmental Microbiology 01/2013; · 5.84 Impact Factor
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ABSTRACT: We report a previously undescribed mechanism for rugose morphotype in Shewanella oneidensis, a research model for investigating redox transformations of environmental contaminants. Bacteria may form smooth or rugose colonies on agar plates. In general, conversion from smooth to rugose colony morphotype is attributed to increased production of exopolysaccharide (EPS). In this work, we discovered that aflagellate S. oneidensis mutants grew into rugose colonies whereas those with non-functional flagellar filaments remained smooth. EPS production was not altered in either case but mutants with rugose morphotype showed significantly reduced exoprotein secretion. The idea that exoproteins at the reduced level correlates with rugosity gained support from smooth suppressor strains of an aflagellate rugose fliD (encoding capping protein) mutant, which restored the exoprotein level to those of the wild type and mutant strains with smooth morphotype. Further analyses revealed that SO1072 (a putative GlcNAc binding protein) was one of highly up-regulated exoproteins in these suppressor strains. Most intriguingly, this study identified a compensatory mechanism of SO1072 to flagellins mediated possibly by c-di-GMP.
Journal of bacteriology 01/2013; · 3.94 Impact Factor
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ABSTRACT: Shewanella oneidensis is a facultative anaerobic γ-proteobacterium possessing remarkably diverse respiratory capacities for reducing various organic and inorganic substrates. As a veteran research model for investigating redox transformations of environmental contaminants the bacterium is well known to be a naturally ampicillin-resistant microorganism. However, in this study we discovered that ampicillin has a significant impact on growth of S. oneidensis. Particularly, cell lysis occurred only with ampicillin at levels ranging from 0.49 to 6.25 µg/ml but not at 50 µg/ml. This phenotype is attributable to insufficient expression of the β-lactamase BlaA. The subsequent analysis revealed that the blaA gene is strongly induced by ampicillin at high (50 µg/ml), but not at low levels (2.5 µg/ml). In addition, we demonstrated that penicillin binding protein 5 (PBP5), the most abundant low molecular weight PBP (LMW PBP), is the only one relevant to β-lactam resistance under the tested conditions. This nonessential PBP, largely resembling its Escherichia coli counterpart in functionality, mediates expression of the blaA gene.
PLoS ONE 01/2013; 8(3):e60460. · 4.09 Impact Factor
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ABSTRACT: Shewanella oneidensis exhibits a remarkable versatility in respiration, which largely relies on its various respiratory pathways. Most of these pathways are composed of secretory terminal reductases and multiple associated electron transport proteins that contain cofactors such as Fe-S, molybdopterin, and NiFe. The majority of these cofactors are inserted enzymatically in the cytoplasm, and thus are substrates of the twin-arginine translocation (Tat) protein export system, which transports fully folded proteins. Using genomic array footprinting, we discovered that loss of TatA or TatC caused a reduction in the growth rate of S. oneidensis under aerobic conditions. Mutational analysis of the predicted Tat substrates revealed that PetA, the Rieske Fe-S subunit of the ubiquinol-cytochrome c reductase, predominantly dictates the aerobic growth defect of tat mutants in S. oneidensis. In addition, evidence is presented that the signal sequence in PetA appears to be resistant to cleavage after the protein is inserted into the cytoplasmic membrane.
PLoS ONE 01/2013; 8(4):e62064. · 4.09 Impact Factor
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ABSTRACT: Shewanella oneidensis exhibits a remarkable versatility in anaerobic respiration, which largely relies on its diverse respiratory pathways. Some of these are expressed in response to the existence of their corresponding electron acceptors (EAs) under aerobic conditions. However, little is known about respiration and the impact of non-oxygen EAs on the physiology of the microorganism when oxygen is present. Here we undertook a study to elucidate the basis for nitrate and nitrite inhibition of growth under aerobic conditions. We discovered that nitrate in the form of NaNO3 exerts its inhibitory effects as a precursor to nitrite at low concentrations and as an osmotic-stress provider (Na(+)) at high concentrations. In contrast, nitrite is extremely toxic, with 25 mM abolishing growth completely. We subsequently found that oxygen represses utilization of all EAs but nitrate. To order to utilize EAs with less positive redox potential, such as nitrite and fumarate, S. oneidensis must enter the stationary phase, when oxygen respiration becomes unfavorable. In addition, we demonstrated that during aerobic respiration the cytochrome bd oxidase confers S. oneidensis resistance to nitrite, which likely functions via nitric oxide (NO).
PLoS ONE 01/2013; 8(4):e62629. · 4.09 Impact Factor
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ABSTRACT: Shewanella inhabit a wide variety of niches in nature and can utilize a broad spectrum of electron acceptors under anaerobic conditions. How they modulate their gene expression to adapt is poorly understood. ArcA, homologue of a global regulator controlling hundreds of genes involved in aerobic and anaerobic respiration in E. coli, was shown to be important in aerobiosis/anaerobiosis of S. oneidensis as well. Loss of ArcA, in addition to altering transcription of many genes, resulted in impaired growth under aerobic condition, which was not observed in E. coli. To further characterize the impact of ArcA loss on gene expression on the level of proteome under aerobic and anaerobic conditions, liquid-chromatography-mass-spectrometry (LC-MS) based proteomic approach was employed. Results show that ArcA loss led to globally altered gene expression, generally consistent with that observed with transcripts. Comparison of transcriptomic and proteomic data permitted identification of 17 high-confidence ArcA targets. Moreover, our data indicate that ArcA is required for regulation of cytochrome c proteins, and the menaquinone level may play a role in regulating ArcA as in E. coli. Proteomic data-guided growth assay revealed that the aerobic growth defect of ArcA mutant is presumably due to impaired peptide utilization.
Proteomics 05/2012; 12(12):1957-69. · 4.43 Impact Factor
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ABSTRACT: Although the pellicle is one of the major growth modes of microorganisms, the metabolic features of pellicle cells and the determinative factors for pellicle formation are largely unknown. In recent years, biofilm development of Shewanella oneidensis, an important model organism for bioremediation studies, has been extensively studied. In this paper, a transcriptional profiling of pellicle cells relative to planktonic cells indicated that cells in pellicles were more metabolically active than the planktonic cells. Most notably, up-transcription of general secretion system proteins and iron/heme uptake and transport proteins was observed in pellicle cells. Unexpectedly, neither the hmuT nor hugA heme transport mutant exhibited a significant defect in pellicle formation. Expectedly, three type I secretion system mutants were severely deficient in pellicle formation, suggesting an essential role of these proteins.
Archives of Microbiology 01/2012; 194(6):473-82. · 1.43 Impact Factor
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ABSTRACT: Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.
PLoS ONE 01/2012; 7(4):e35099. · 4.09 Impact Factor
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ABSTRACT: We have previously illustrated the nitrate/nitrite respiratory pathway of Shewanella oneidensis, which is renowned for its remarkable versatility in respiration. Here we investigated the systems regulating the pathway with a reliable approach which enables characterization of mutants impaired in nitrate/nitrite respiration by guaranteeing biomass. The S. oneidensis genome encodes an Escherichia coli NarQ/NarX homolog SO3981 and two E. coli NarP/NarL homologs SO1860 and SO3982. Results of physiological characterization and mutational analyses demonstrated that S. oneidensis possesses a single two-component system (TCS) for regulation of nitrate/nitrite respiration, consisting of the sensor kinase SO3981(NarQ) and the response regulator SO3982(NarP). The TCS directly controls the transcription of nap and nrfA (genes encoding nitrate and nitrite reductases, respectively) but regulates the former less tightly than the latter. Additionally, phosphorylation at residue 57 of SO3982 is essential for its DNA-binding capacity. At the global control level, Crp is found to regulate expression of narQP as well as nap and nrfA. In contrast to NarP-NarQ, Crp is more essential for nap rather than nrfA.
PLoS ONE 01/2012; 7(12):e51643. · 4.09 Impact Factor
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ABSTRACT: Tetrahydrofuran (THF) is a toxic and carcinogenic compound that is commonly released from pharmaceutical, chemical and related industry wastewater. Currently, the effects of THF contamination on wastewater are unknown and a better understanding of THF toxicity toward biological processes in wastewater treatment is critical. In this study, we firstly investigated the toxic effects of THF on enzymatic activity and the microbial diversity in activated sludge from a sequencing batch reactor during long-term exposure to 10 mM THF. The activity of five enzymes (catalase, dehydrogenase, urease, phosphatase and protease) was remarkably decreased in the presence of 10 mM THF during a period of 85 days. Of these five affected enzymes, dehydrogenase activity was close to detection level limits and was nearly completely inhibited. Analysis of the microbial community demonstrated that THF, at a concentration of 10 mM, altered the distribution of microbes within the community and significantly decreased microbial diversity during long-term contamination, according to denaturing gradient gel electrophoresis (DGGE) analysis. The fraction of Actinobacteria increased in the community, while the fraction of Proteobacteria significantly decreased after THF exposure.
Ecotoxicology 08/2011; 21(1):56-65. · 2.36 Impact Factor
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ABSTRACT: Two closely related, Gram-stain-negative, rod-shaped, spore-forming strains, B27(T) and F6-B70, were isolated from soil samples of Tianmu Mountain National Natural Reserve in Zhejiang, China. Phylogenetic analysis based on 16S rRNA gene and rpoB sequences indicated that the isolates were members of the genus Paenibacillus. Both isolates were closely related to Paenibacillus ehimensis IFO 15659(T), Paenibacillus elgii SD17(T) and Paenibacillus koreensis YC300(T) (≥ 95.2 % 16S rRNA gene sequence similarity). DNA-DNA relatedness between strain B27(T) and P. ehimensis DSM 11029(T), P. elgii NBRC 100335(T) and P. koreensis KCTC 2393(T) was 21.2, 28.6 and 16.8 %, respectively. The major cellular fatty acids of strains B27(T) and F6-B70 were anteiso-C(15 : 0) and iso-C(15 : 0). The cell wall contained meso-diaminopimelic acid. The two isolates differed from their closest neighbours in terms of phenotypic characteristics and cellular fatty acid profiles (such as variable for oxidase, negative for methyl red test, unable to produce acid from d-fructose and glycogen and relatively higher amounts of iso-C(15 : 0) and lower amounts of C(16 : 0) and iso-C(16 : 0)). Strains B27(T) and F6-B70 represent a novel species of the genus Paenibacillus, for which the name Paenibacillus tianmuensis sp. nov. is proposed. The type strain is B27(T) ( = DSM 22342(T) = CGMCC 1.8946(T)).
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 05/2011; 61(Pt 5):1133-7. · 2.11 Impact Factor
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ABSTRACT: EnvZ and OmpR constitute the bacterial two-component signal transduction system known to mediate osmotic stress response in a number of gram-negative bacteria. In an effort to understand the mechanism through which Shewanella oneidensis senses and responds to environmental osmolarity changes, structure of the ompR-envZ operon was determined with Northern blotting assay and roles of the EnvZ/OmpR two-component system in response to various stresses were investigated with mutational analysis, quantitative reverse transcriptase PCR (qRT-PCR), and phenotype microarrays. Results from the mutational analysis and qRT-PCR suggested that the EnvZ/OmpR system contributed to osmotic stress response of S. oneidensis and very likely engaged a similar strategy employed by E. coli, which involved reciprocal regulation of two major porin coding genes. Additionally, the ompR-envZ system was also found related to cell motility. We further showed that the ompR-envZ dependent regulation of porin genes and motility resided almost completely on ompR and only partially on envZ, indicating additional mechanisms for OmpR phosphorylation. In contrast to E. coli lacking ompR-envZ, however, growth of S. oneidensis did not show a significant dependence on ompR-envZ even under osmotic stress. Further analysis with phenotype microarrays revealed that the S. oneidensis strains lacking a complete ompR-envZ system displayed hypersensitivities to a number of agents, especially in alkaline environment. Taken together, our results suggest that the function of the ompR-envZ system in S. oneidensis, although still connected with osmoregulation, has diverged considerably from that of E. coli. Additional mechanism must exist to support growth of S. oneidensis under osmotic stress.
PLoS ONE 01/2011; 6(8):e23701. · 4.09 Impact Factor
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ABSTRACT: A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.
Journal of Industrial Microbiology 01/2011; 38(9):1507-13. · 1.80 Impact Factor
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ABSTRACT: The shewanellae are ubiquitous in aquatic and sedimentary systems that are chemically stratified on a permanent or seasonal basis. In addition to their ability to utilize a diverse array of terminal electron acceptors, the microorganisms have evolved both common and unique responding mechanisms to cope with various stresses. This paper focuses on the response and adaptive mechanism of the shewanellae, largely based on transcriptional data.
International Journal of Microbiology 01/2011; 2011:863623.
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ABSTRACT: Shewanella oneidensis is a highly motile organism by virtue of a polar flagellum. Unlike most flagellated bacteria, it contains only one major chromosome segment encoding the components of the flagellum with the exception of the motor proteins. In this region, three genes encode flagellinsaccording to the original genome annotation. However, we find that only flaA and flaB encode functional filament subunits. Although these two genesare under the control of different promoters, they are actively transcribed and subsequently translated, producing a considerable number of flagellin proteins. Additionally, both flagellins are able to interact with their chaperon FliS and are subjected to feedback regulation. Furthermore, FlaA and FlaB are glycosylated by a pathwayinvolving a major glycosylating enzyme,PseB, in spite of the lack of the majority of theconsensus glycosylation sites. In conclusion, flagellar assembly in S. oneidensis has novel features despite the conservation of homologous genes across taxa.
PLoS ONE 01/2011; 6(6):e21479. · 4.09 Impact Factor
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ABSTRACT: Although solid surface-associated biofilm development of S. oneidensis has been extensively studied in recent years, pellicles formed at the air-liquid interface are largely overlooked. The goal of this work was to understand basic requirements and mechanism of pellicle formation in S. oneidensis.
We demonstrated that pellicle formation can be completed when oxygen and certain cations were present. Ca(II), Mn(II), Cu(II), and Zn(II) were essential for the process evidenced by fully rescuing pellicle formation of S. oneidensis from the EDTA treatment while Mg (II), Fe(II), and Fe(III) were much less effective. Proteins rather than DNA were crucial in pellicle formation and the major exopolysaccharides may be rich in mannose. Mutational analysis revealed that flagella were not required for pellicle formation but flagellum-less mutants delayed pellicle development substantially, likely due to reduced growth in static media. The analysis also demonstrated that AggA type I secretion system was essential in formation of pellicles but not of solid surface-associated biofilms in S. oneidensis.
This systematic characterization of pellicle formation shed lights on our understanding of biofilm formation in S. oneidensis and indicated that the pellicle may serve as a good research model for studying bacterial communities.
BMC Microbiology 11/2010; 10:291. · 3.04 Impact Factor
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ABSTRACT: Paenibacillus sp. F6-B70 was selected from several dozens of isolates with activity against methicillin-resistant Staphylococcus aureus using a 16S rDNA-based screening method. F6-B70 contained polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters in its genome revealed by PCR amplification of conserved adenylation and ketosynthase (KS) domains. Phylogenetic data suggested that the strain hosts trans-AT PKSs and their product may be a branched molecule. An antibiotic was subsequently isolated from the methanol extract of F6-B70 cells. The molecular formula of the antibiotic was deduced to be C(33) H(50) NaO(6) ([M + Na](+) , m/z 565.3505) by analysis of electrospray ionization mass spectral data. Elucidation of the structure by nuclear magnetic resonance and infrared spectroscopy revealed that the active compound, paenimacrolidin (PAM), was a novel 22-membered macrolide with side-chains. The new antibiotic, mainly as a bacteriostatic agent, inhibits a couple of multidrug-resistant Staphylococcus sp. strains. The antibiotic capacity of PAM was compromised by its instability, which can be overcome significantly with addition of an anti-oxidant. To our knowledge, this is the first report of the isolation of an active macrolide from paenibacilli, which may be a promising source of novel antibiotics.
Microbial Biotechnology 08/2010; 4(4):491-502. · 2.53 Impact Factor
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Haichun Gao,
Soumitra Barua,
Yili Liang,
Lin Wu,
Yangyang Dong,
Samantha Reed,
Jingrong Chen,
Dave Culley,
David Kennedy,
Yunfeng Yang,
Zhili He,
Kenneth H Nealson,
James K Fredrickson,
James M Tiedje,
Margaret Romine,
Jizhong Zhou
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ABSTRACT: Shewanella are renowned for their ability to utilize a wide range of electron acceptors (EA) for respiration, which has been partially accredited to the presence of a large number of the c-type cytochromes. To investigate the involvement of c-type cytochrome proteins in aerobic and anaerobic respiration of Shewanella oneidensis Mr -1, 36 in-frame deletion mutants, among possible 41 predicted, c-type cytochrome genes were obtained. The potential involvement of each individual c-type cytochrome in the reduction of a variety of EAs was assessed individually as well as in competition experiments. While results on the well-studied c-type cytochromes CymA(SO4591) and MtrC(SO1778) were consistent with previous findings, collective observations were very interesting: the responses of S. oneidensis Mr -1 to low and highly toxic metals appeared to be significantly different; CcoO, CcoP and PetC, proteins involved in aerobic respiration in various organisms, played critical roles in both aerobic and anaerobic respiration with highly toxic metals as EA. In addition, these studies also suggested that an uncharacterized c-type cytochrome (SO4047) may be important to both aerobiosis and anaerobiosis.
Microbial Biotechnology 07/2010; 3(4):455-66. · 2.53 Impact Factor
