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The Determination of Molecular Weight of Bacterial Genome DNA from Renaturation Rates

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Abstract

A new method is described for the determination of the total molecular weight of haploid genome DNA. It is based on initial optical renaturation rate measurements of precisely known concentrations of fragmented DNA. The theoretical basis of the measurement is presented. The actual state of replication of the genome has little effect. The exact conditions and the effects of DNA concentration, renaturation time, size of DNA fragments, buffer concentration, optimal temperature and % (G + C) have been determined. Several types of bacterial DNA of known genome size were included as a control. As an application, the DNA genome size have been determined of 40 different bacteria. The method appears to offer advantages by its simplicity, rapidity and reproducibility.

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... Several bacterial media were used to check the growth, such as Luria-Bertani agar (LB agar; Oxoid), Nutrient agar (NA, Difco), R2A (Difco), Tryptone soya agar (TSA; Oxoid), and MacConkey agar (Oxoid) at 28 °C. Growth at various temperatures (4,10,15,20,25,28,30,35, 40° and 45 °C) and numerous pH conditions (pH 4.0-10.0, 0.5 pH unit interval) were evaluated in R2A broth after 7 days of incubation at 28 °C. ...
... DNA-DNA hybridization tests were performed for strain MAH-34 T and its closely related reference strains. The optimum renaturation temperature (35 °C) was calculated as [(0.51 × G + C content) + 47]-36 [25], where 36 °C is the correction for the presence of 50% formamide [26]. Hybridization was done with five replications for each 1 3 sample. ...
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A silver nanoparticle (AgNP) producing, Gram stain-positive, aerobic, motile, and rod-shaped novel bacterial strain, designated as MAH-34 T was isolated from rhizospheric soil of magnolia tree. The colonies were creamy white, smooth, circular, and 0.9–2.0 mm in diameter when grown on R2A agar. Strain MAH-34 T was found to be able to grow at 10–37 °C, at pH 6.0–9.5, and at 0–1% NaCl. The strain showed activity for both catalase, and oxidase tests, and was able to rapid synthesis of AgNPs. The TEM image revealed the spherical shape of biosynthesized AgNPs, and the size was 5 to 15 nm. Based on 16S rRNA gene sequence comparisons, the isolate was shown to be a member of genus Paenibacillus, and the close type strains were Paenibacillus chondroitinus DSM 5051 T (98.3%), Paenibacillus aceris KUDC4121T (98.2%), Paenibacillus nebraskensis JJ-59 T (97.8%), Paenibacillus alginolyticus DSM 5050 T (97.6%), Paenibacillus ferrarius CY1T (97.4%), Paenibacillus frigoriresistens YIM 016 T (97.3%), and Paenibacillus pocheonensis Gsoil 1138 T (97.3%). Strain MAH-34 T had a genome size of 8,647,010 bp. The genomic G + C content was 46.0 mol %. The major isoprenoid quinone was determined as menaquinone-7 (MK-7). The major cellular fatty acids were determined as C15:0 anteiso, and C16:0 iso. Based on the DNA-DNA hybridization results, genotypic analysis, chemotaxonomic, and physiological data, strain MAH-34 T represents a novel species, for which the name Paenibacillus anseongense sp. nov. is proposed, with MAH-34 T as the type strain (= KACC 19974 T = CGMCC1.16610 T).
... MEGA 7 software was used to construct a phylogenetic tree by bootstrap analysis with 1000 replications (Felsenstein 1985). The taxonomic relationships between strain KVB24 T and its close phylogenetic neighbors were examined using the DNA-DNA hybridization method as previously described (De Ley et al. 1970;Gillis et al. 1970) and an optimized procedure described previously (Loveland-Curtze et al. 2011). The sequence of Rhodanobacter lindaniclasticus RP5557 T (AF039167) was used as an out-group. ...
... DNA-DNA hybridization was performed to analyse the relationships of the novel isolate with related taxa based on the thermal denaturation principles and equations (De Ley et al. 1970, Gillis et al. 1970) and an optimized procedure described previously (Loveland-Curtze et al. 2011). For genomic characterization of strain KVB24 T , we performed DNA-DNA hybridization analyses between the strain KVB24 T and L. dokdonensis DS-58 T using the fluorimetric method. ...
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A novel bacterial strain, designated KVB24T, was isolated from sea-water of Busan Harbour in South Korea. Cells of strain KVB24T were Gram-stain negative, aerobic, rod shaped and non-motile. Strain KVB24T grew optimally at 25–28 °C and pH 6.5–7.0. Based on 16S rRNA gene sequence analysis, strain KVB24T was shown to belong to the genus Lysobacter within the class Gammaproteobacteria and to be closely related to Lysobacter dokdonensis DS-58T, Lysobacter hankyongensis KTce-2T and Lysobacter niastensis GH41-7T. DNA–DNA relatedness between strain KVB24T and its current closest relative was below 70%. The predominant fatty acids of strain KVB24T were iso-C11:0, iso-C11:0 3-OH, iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0 and summed feature 9 comprising (iso-C17:1 ω9c and/or 10 methyl C16:0); the prominent isoprenoid was Q-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The G + C content of genomic DNA from strain KVB24T was determined to be 67.5 mol%. Based on the phenotypic, genotypic and chemotaxonomic analyses, strain KVB24T represents a novel species of the genus Lysobacter, for which the name Lysobacter caseinilyticus sp. nov. is proposed. The type strain is KVB24T (= KACC19816T = JCM32879T).
... Genomic DNA of seven references was used for calibration (Ausubel et al. 1995). DNA-DNA hybridization was performed to analyse the relationships of the novel isolate with related taxa based on the thermal denaturation principles and equations of De and Gillis et al. (1970) and an optimized procedure described by Loveland-Curtze et al. (2011). These experiments were independently performed in triplicate. ...
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An aerobic, Gram-stain-negative, bright yellow-pigmented, oxidase and catalase-positive, non-motile, non-spore forming, rod-shaped strain designated DMN11T was isolated from the soil of crossroads of Jeju Island in South Korea. Colonies were circular, bright yellow-pigmented and smooth with regular edges and measured approximately 1–2 mm in diameter. Flexirubin-type pigments were absent. Phylogenetic tree analysis based on the 16SrRNA gene sequence revealed that the strain DMN11T formed a lineage within the family Flavobacteriaceae of the phylum Bacteroidetes, and it was the most closely related to Flavobacterium suzhouense XIN-1T and Flavobacterium hauense BX12T (98.6% and 98.2% similarity, respectively). The major isoprenoid quinone was MK-6. The major fatty acids were summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), iso-C15:0 and iso-C15:0 3OH. The polar lipid profile of the strain DMN11T showed the presence of phosphatidylethanolamine (PE) as major lipid. The DNA G+C content was 35.3 mol%, as determined by the thermal denaturation method. The mean levels of DNA–DNA relatedness of the strain DMN11T with F. suzhouense XIN-1T and F. hauense BX12T were 20.5% and 29.2%, respectively. Thus, the data accumulated in this study support the suggestion that the strain DMN11T is considered to represent a novel species of the genus Flavobacterieum, for which the name Flavobacterium edaphi sp. nov. is proposed. The type strain is DMN11T (= KCTC 62114T = JCM 32372T).
... DNA suspended in 2ÂSSC and 1 : 10000 SYBR Green was used for the analysis. DNA denaturation at 95.0 C was carried out followed by reassociation at the optimum temperature of 67.0 C. Fluorescence readings were taken every 10 s throughout the protocol and relative binding ratio was calculated according to the methods of De Ley et al. [40] and Gillis et al. [41]. ...
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A curved-rod-shaped bacterium was isolated from a marine (100 m depth) water sample collected from Bay of Bengal, Visakhapatnam, India. Strain NIO-S14T, was Gram-stain-negative, motile and pale-yellow. NIO-S14T was able to grow aerobically and anaerobically and could utilize a number of organic substrates. Major fatty acids were C12 : 0, iso-C13 : 0, C14 : 0, iso-C15 : 0, C16 : 0 and C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3). NIO-S14T contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminophospholipids and six unidentified lipids as polar lipids. The DNA G+C content of NIO-S14T was 47.9 mol%. The 16S rRNA gene sequence comparisons indicated that the isolate represented a member of the family Shewanellaceae within the class Gammaproteobacteria. According to the results of 16S rRNA gene sequence analysis, NIO-S14T was closely related to Shewanella coralliiwith a pair-wise sequence similarity of 99.26 %. On the basis of the sequence comparison, NIO-S14T clustered with Shewanella coralliiand together they clustered with Shewanella mangroviand seven other species of the genus Shewanella but were distantly related. DNA-DNA hybridization between NIO-S14T and Shewanella corallii DSM 21332Trevealed a relatedness of 35 %. Distinct morphological, physiological and genotypic differences from these previously described taxa supported the classification of NIO-S14T as a representative of a novel species of the genus Shewanella, for which the name Shewanellasubmarina sp. nov. is proposed. The type strain of Shewanellasubmarina is NIO-S14T (=MTCC 12524T=KCTC 52277T=LMG 30752T).
... DNA-DNA hybridization experiments were performed for strain MAH-12 T and its closely related reference strain Paenibacillus barengoltzii KACC 15270 T . The optimum renaturation temperature (37 °C) was calculated as [(0.51 × G + C content) + 47] − 36 [12], where 36 °C is the correction for the presence of 50% formamide [24]. Hybridization was performed with five replications for each sample. ...
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A Gram-stain positive, aerobic, motile, rod-shaped, and ginsenoside Rd producing novel bacterial strain, designated as MAH-16T, was isolated from soil sample of a vegetable garden and was characterized by using a polyphasic approach. The colonies were beige color, smooth, circular, and 0.3–0.7 mm in diameter when grown on tryptone soya agar for 3 days. Strain MAH-16T can grow at 20–40 °C temperature, at pH 5.0–7.0 and at 0–1% NaCl. Cell growth occurs on nutrient agar, R2A agar, tryptone soya agar, and Luria–Bertani agar but not on MacConkey agar. The strain was positive for both catalase and oxidase test. The novel strain rapidly synthesized ginsenoside Rd from major ginsenoside Rb1. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Paenibacillus and was most closely related to Paenibacillus barengoltzii SAFN-016T (97.1%), Paenibacillus faecis 656.84T (96.7%), and Paenibacillus konsidensis LBYT (96.2%). In DNA–DNA hybridization tests, the DNA relatedness between strain MAH-16T and its closest phylogenetic neighbor was below 45.0%. The genomic DNA G + C content of isolated strain was determined to be 52.0 mol% and the predominant isoprenoid quinine was menaquinone-7 (MK-7). The major fatty acids were identified as anteiso-C15:0. The genetic characteristics in combination with chemotaxonomic and physiological data demonstrated that strain MAH-16T represented a novel species within the genus Paenibacillus, for which the name Paenibacillus horti sp. nov. is proposed, with MAH-16T as the type strain (=KACC 19299T = CGMCC1.16487T).
... Genomic DNA of seven references was used for calibration (Ausubel et al. 1995). DNA-DNA hybridization was performed to analyse the relationships of the novel isolate with related taxa based on the thermal denaturation principles and equations of De and Gillis et al. (1970) and an optimized procedure described by Loveland-Curtze et al. (2011). These experiments were independently performed in triplicate. ...
... DNA G ? C mol% and DNA-DNA hybridization of genomic analysis Total genomic DNA was extracted and purified to evaluate the G ? C mol% content and DNA-DNA hybridizations, following the method described by Ausubel et al. (1995). The DNA G ? C content of strains D10 T and U8 T were determined using a simple fluorimetric method (Gonzalez and Saiz-Jimenez 2002) with SYBR Green 1 (Life Technologies, Waltham, USA) and a real-time PCR thermocycler (Roter-Gene Q, Qiagen, Hilden, Germany The DNA-DNA hybridization analysis was performed to determine the relatedness of the novel isolates and related taxa based on denaturation principles and the equations introduced by De and Gillis et al. (1970) as well as an optimised procedure delineated by Loveland-Curtze et al. (2011). The DNA-DNA hybridization values were assessed with the fluorimetric method using real-time PCR. ...
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Two bacterial strains, designated D10T and U8T, were isolated from soil samples from the Dong-angyeong cave and Geommeolle wharf sea-coast, Udo-Island, Jeju, South Korea. Both novel bacterial strains are yellow-pigmented, Gram-stain negative, motile by means of monotrichous flagella, short rod shaped and strictly aerobic. A phylogenetic tree was reconstructed based on their 16S rRNA gene sequences, which indicated that these two strains belong to the genus Lysobacter within the family Xanthomonadaceae. Strain D10T showed high 16S rRNA gene sequence similarities with Lysobacter humi FJY8T (99.0%), Lysobacter xinjiangensis RCML-52T (98.9%) and Lysobacter mobilis 9NM-14T (97.2%), whereas strain U8T showed high sequence similarities to L. mobilis 9NM-14T (97.9%), L. xinjiangensis RCML-52T (97.8%), L. humi FJY8T (97.5%) and Lysobacter bugurensis ZLD-29T (97.1%). The 16S rRNA gene sequence similarity between D10T and U8T was 97.0%. Strain D10T showed low DNA–DNA relatedness to U8T (57.7 ± 3.4%), L. humi FJY8T (48.8 ± 4.3%), L. xinjiangensis RCML-52T (60.1 ± 2.4%) and L. mobilis 9NM-14T (55.9 ± 1.9%). The level of DNA–DNA relatedness for strain U8T with respect to D10T, L. mobilis 9NM-14T, L. xinjiangensis RCML-52T, L. humi FJY8T, and L. bugurensis ZLD-29T was 55.5 ± 0.5%, 54.5 ± 2.1%, 58.1 ± 0.8%, and 51.9 ± 3.4%, respectively. The major polar lipids for both strains were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids for both strains were identified as iso-C15:0, iso-C16:0 and summed feature 9 (iso-C17:1 ω9c/C16:0 10-methyl), and ubiquinone (Q-8) as the only isoprenoid quinone for both strains. The DNA G + C contents of the strains D10T and U8T were determined to be 70.2 mol% and 70.6 mol%. On the basis of phenotypic, genotypic, chemotaxonomic, and phylogenetic analysis, both strains D10T and U8T represent a novel species in the genus Lysobacter, for which the names Lysobacter helvus sp. nov. and Lysobacter xanthus sp. nov. are proposed, respectively. The type strain of L. helvus is D10T (= KCTC 62111T = JCM 32364T) and the type strain of L. xanthus is U8T (= KCTC 62112T = JCM 32365T).
... G?C content) ? 47] -36 (Gillis et al. 1970), where 36°C is the correction for the presence of 50% formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. ...
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A novel Gram-staining negative, yellow-pigmented, non-motile, aerobic and rod-shaped bacterium, designated MAH-11T, was isolated from rhizosphere of Pinus koraiensis and was characterised by using a polyphasic taxonomic approach. The colonies were smooth, circular and 0.3–1.0 mm in diameter when grown on R2A agar for 3 days. The strain was positive for both catalase and oxidase tests. Optimum growth temperature and pH were 28–30 °C and 7.0, respectively. Cell growth occurs on R2A agar, nutrient agar, Luria–Bertani agar and tryptone soya agar but not on MacConkey agar. The novel strain was found to be able to hydrolyse esculin but not casein, gelatin, starch, l-tyrosine, DNA, l-arginine, urea, Tween 20 and Tween 80. On the basis of 16S rRNA gene sequence analysis, strain MAH-11T belongs to the genus Sphingobium and is closely related to Sphingobium quisquiliarum P25T (98.1%), Sphingobium vermicomposti VC-230T (97.8%), Sphingobium mellinum WI4T (97.5%), Sphingobium barthaii KK22T (97.2%) and Sphingobium fuliginis TKPT (97.2%). In DNA–DNA hybridization tests, the DNA relatedness values between strain MAH-11T and its close phylogenetic neighbors were below 45.0%. The DNA G+C content was 64.5 mol% and the predominant respiratory quinone was identified as ubiquinone-10. The major cellular fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16:0. The DNA–DNA hybridization results in combination with chemotaxonomic and physiological data demonstrated that strain MAH-11T represents a novel species within the genus Sphingobium, for which the name Sphingobium chungangianum is proposed. The type strain is MAH-11T (= KACC 19836T = CGMCC 1.13749T).
... The optimum renaturation temperature (33°C) was calculated as [(0.51 9 G ? C content) ? 47]-36 (Gillis et al. 1970), where 36°C is the correction for the presence of 50% formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. ...
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A Gram-stain negative, aerobic, non-motile and rod-shaped novel bacterial strain, designated as MAH-5T, was isolated from a road-side soil sample and was characterised by using a polyphasic taxonomic approach. The colonies were observed to be yellowish orange in colour, smooth, circular and 0.3–0.7 mm in diameter when grown on nutrient agar for 2 days. Strain MAH-5T was found to be able to grow at 15–35 °C and at pH 4.0–8.0. The strain was observed to be positive for both the catalase and oxidase tests. Cells were found to be able to hydrolyse aesculin, gelatin and starch. By 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Mucilaginibacter and to be closely related to Mucilaginibacter panaciglaebae BXN5-31T (98.35%), Mucilaginibacter soyangensis HME6664T (97.82%), Mucilaginibacter antarcticus S14-88T (97.49%) and Mucilaginibacter ximonensis XM-003T (97.06%). In DNA–DNA hybridization tests, the DNA relatedness values between strain MAH-5T and its close phylogenetic neighbors were below 45.0%. The genomic DNA G + C content of strain MAH-5T was determined to be 41.5 mol% and the predominant isoprenoid quinine was identified as MK-7. The major fatty acids were identified as C15:0 iso and summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c). The genetic characteristics, in combination with chemotaxonomic and physiological data, demonstrated that the isolated strain MAH-5T represents a novel species within the genus Mucilaginibacter, for which the name Mucilaginibacter formosus sp. nov. is proposed, with MAH-5T as the type strain (= KACC 19291T = CGMCC1.16489T).
... The DNA-DNA hybridization analysis was performed to determine the relatedness of the novel isolate to related taxa, based on denaturation principles, equations introduced by De and Gillis et al. (1970), as well as an optimised procedure delineated by Loveland-Curtze et al. (2011). The DNA-DNA hybridization values were assessed using real-time PCR, with a fluorimetric method. ...
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A Gram-stain positive, short rod-shaped, aerobic, motile by means of gliding, yellow-pigmented actinobacterium, designated strain DD4aT, was isolated from dry yellow foxtail. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DD4aT is closely related to Amnibacterium soli MB78T (98.4% similarity), Amnibacterium kyonggiense KSL51201-037T (98.2%) and Amnibacterium endophyticum 1T4Z-3T (97.43%). Strain DD4aT forms yellow colonies on R2A agar medium. The peptidoglycan was found to contains diaminopimelic acid (which is a diagnostic cell wall diamino acid), alanine, glutamic acid and lysine. The polar lipids diphosphatidylglycerol, phosphatidylglycerol, six unidentified glycolipids and an unidentified polar lipid were found to be present in strain DD4aT. The major cellular fatty acids anteiso-C15:0 (42.9%) and iso-C16:0 (34.6%) were found in strain DD4aT. The predominant respiratory quinones were found to be MK-11 and MK-12. The DNA G+C content of strain DD4aT is 73.9 mol%. DNA–DNA relatedness of strain DD4aT with A. soli MB78T, A. kyonggiense KSL51201-037T, and A. endophyticum 1T4Z-3T were 53.3% (± 1.1%), 47.0% (± 0.5%), and 47.9% (± 0.9%), respectively. The digital DNA–DNA hybridisation and average nucleotide identity values between strain DD4aT and A. kyonggiense KSL51201-037T were determined to be 26.1% and 82.7%. On the basis of phenotypic, genotypic, chemotaxonomic and phylogenetic analysis, DD4aT represents a novel member of the genus Amnibacterium, for which the name Amnibacterium setariae sp. nov., is proposed. The type strain of Amnibacterium setariae is DD4aT (= KACC 19817T = JCM 32878T).
... The DNA G + C content of strain IP7 T was determined using a simple fluorimetric method (Gonzalez and Saiz-Jimenez, 2002) with SYBR Green 1 (Life Technologies) and a real-time PCR thermocycler (Roter-Gene Q, Qiagen T were used for calibration. DNA-DNA hybridization analysis was conducted to determine the relatedness of the novel isolates and correlated taxa based on denaturation principles and the equations introduced by De Ley et al. (1970) and Gillis et al. (1970) as well as an optimized procedure delineated by Loveland-Curtze et al. (2011). DNA-DNA hybridization values were assessed with the fluorimetric method using real-time PCR, and Aestuariibaculum suncheonense SC17 T selected as the closest relative to the novel strain. ...
Article
A Gram-negative, non-motile, aerobic bacterium, designated strain IP7T, was isolated from seawater at the shore of the Incheon Eulwang-ri beach, South Korea. Cells of strain IP7T are straight or slightly rod-shaped and colonies are round, convex and orange-yellow. Strain IP7T is flexirubin-negative, mild halophile, catalase-and oxidase-positive, and produces a yellow-orange carotenoid pigment. Growth is optimal at 30°C, pH 7–9, and 2.0–4.0% NaCl (w/v). On the basis of 16S rRNA gene sequence similarity, strain IP7T is affiliated with genus Aestuariibaculum in the family Flavobacteriaceae, the closest relative being Aestuariibaculum suncheonense SC17T (98.3% sequence similarity). The DNA G + C content of the novel strain is 37.4 mol%. The only quinone is MK-6 menaquinone. Iso-branched C15:0, iso-branched C15:1 G, and iso-branched C17:0 3-OH are major fatty acids. The major polar lipids are phosphatidylethanolamine, an unidentified aminoglycolipid and two unidentified glycolipids. The DNA-DNA hybridization value of strain IP7T with Aestuariibaculum suncheonense SC17T is 28.87%. Based on the collective DNA-DNA hybridization, biochemical, phylogenetic and physiological data, we report a novel species of the genus Aestuariibaculum for which the name Aestuariibaculum marinum sp. nov. is proposed. The type strain is IP7T (= KCTC 52521T = JCM 31725T).
... DNA-DNA hybridization experiments were performed between strain MAH-2 T and closely related type strains of genus Microvirga. The optimum renaturation temperature (42 °C) was calculated as [(0.51 × G + C content) + 47] − 36 (Gillis et al. 1970), where 36 °C is the correction for the presence of 50% formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. ...
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A Gram-stain negative, aerobic, non-motile, and rod-shaped novel bacterial strain, designated MAH-2T, was isolated from a soil sample of rose garden and was characterized using a polyphasic approach. The colonies were light pink color, smooth, circular and 0.2–0.6 mm in diameter when grown on nutrient agar for 3 days. Strain MAH-2T grows at 15–40 °C (optimum growth temperature 30 °C), at pH 5.0–7.0 (optimum growth pH 6.5) and at 0–2% NaCl (optimum 0-0.5%). Cell growth occurs on nutrient agar and R2A agar but not on tryptone soya agar, luria–bertani agar and MacConkey agar. The strain was positive for both catalase and oxidase tests. The strain was able to synthesis of silver nanoparticles. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Microvirga and was most closely related to Microvirga soli R491T (96.7% sequence similarity), Microvirga subterranea Fail4T (96.4%), Microvirga guangxiensis 25BT (96.0%) and Microvirga aerophila 5420S-12T (95.9%). The genomic DNA G + C content of isolated strain was determined to be 62.5 mol% and the predominant isoprenoid quinone was Q-10. The major fatty acids were identified as summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c. On the basis of these phenotypic, genotypic, and chemotaxonomic studies and DNA–DNA hybridization results, the isolated strain MAH-2T represents a novel species, for which the name Microvirga rosea sp. nov. is proposed, with MAH-2T as the type strain (= KACC 19290T = CGMCC1.16488T).
... DNA-DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989) with modifications (Stabili et al. 2008), using photobiotin-labelled DNA probes and microdilution wells. The optimum renaturation temperature (33 °C) was calculated as [(0.51 × G + C content) + 47] − 36 (Gillis et al. 1970), where 36 °C is the correction for the presence of 50% formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. ...
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A Gram-stain negative, aerobic, motile and rod-shaped novel bacterial strain, designated MAH-3T, was isolated from soil sample of a rice field. The colonies were orange pigmented, smooth, circular and 0.4–0.9 mm in diameter when grown on R2A agar for 3 days. Strain MAH-3T was found to grow at 10–40 °C (optimum 30 °C), pH 6.0–8.0 (optimum 7.0) and in the presence of 0–1.0% NaCl (optimum 0%). Cell growth occurs on R2A agar and Luria–Bertani agar, but not on nutrient agar, tryptone soya agar and MacConkey agar. Cells were positive for catalase test but negative for oxidase test. Cells were able to hydrolyze casein, gelatin, DNA and Tween 80. 16S rRNA gene sequence analysis revealed that strain MAH-3T was most closely related to the genus Fluviicola and exhibited the highest sequence similarity to Fluviicola hefeinensis MYL-8T (97.4%), Fluviicola taffensis RW262T (96.2%) and Fluviicola kyonggii CA-1T (95.6%). Strain MAH-3T had a genome size of 4,271,694 bp and the genomic DNA G + C content was determined to be 41.7 mol%. The genome contained 19 contigs encoded by 3,664 protein-coding genes with 34 tRNA and 4 rRNA genes. The genomic ANI value between strain MAH-3T and one of the closely related type strains, F. taffensis DSM 16823T was 76.2%. The predominant isoprenoid quinone of isolated strain MAH-3T was menaquinone-6 (MK-6). The major fatty acids were identified as C15:0 iso, C15:0 2OH and C17:0 iso 3OH. On the basis of these phenotypic, genotypic and chemotaxonomic studies and DNA–DNA hybridization results, the isolated strain MAH-3T represents a novel species, for which the name Fluviicola chungangensis sp. nov. is proposed, with MAH-3T as the type strain (= KACC 19742T = CGMCC 1.13750T).
... The optimum renaturation temperature (33°C) was calculated as [(0.51 9 G?C content) ? 47] -36 (Gillis et al. 1970), where 36°C is the correction for the presence of 50% formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. ...
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A gram-stain negative, aerobic, non-motile and rod-shaped novel bacterial strain, designated MAH-19T, was isolated from bark of Pinus koraiensis. The colonies were observed to be light pink coloured, smooth, circular and 0.3–0.7 mm in diameter when grown on R2A agar for 2 days. Strain MAH-19T was found to be able to grow at 10–35 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–0.5% NaCl (optimum 0%). Cell growth occurs on nutrient agar and R2A agar. The strain was found to be positive for both catalase and oxidase tests. Cells are able to hydrolyse aesculin and Tween 20, but not casein, gelatin, starch, l-tyrosine, DNA, l-arginine, urea or Tween 80. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Mucilaginibacter and to be closely related to Mucilaginibacter panaciglaebae BXN5-31T (97.4% similarity), Mucilaginibacter antarcticus S14-88T (97.2%) and Mucilaginibacter ximonensis XM-003T (97.1%). In DNA–DNA hybridization tests, the DNA relatedness between strain MAH-19T and its close phylogenetic neighbours was below 45.0%. The novel strain MAH-19T has a draft genome size of 5,335,442 bp (14 contigs), annotated with 4963 protein-coding genes, 44 tRNA and 6 rRNA genes. The genomic DNA G+C content was determined to be 42.7 mol%. The predominant isoprenoid quinone of strain MAH-19T was identified as MK-7. The major fatty acids were identified as C15:0 iso and summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c). The DNA–DNA hybridization results and results of the genotypic analysis, in combination with chemotaxonomic and physiological data, demonstrated that strain MAH-19T represents a novel species within the genus Mucilaginibacter, for which the name Mucilaginibacter corticis sp. nov. is proposed, with MAH-19T (= KACC 19745T = CGMCC1.13657T) as the type strain.
... DNA suspended in 2× SSC (Saline Sodium citrate) was used for the analysis. The re-association of DNA was carried out at optimum re-association temperature 71°C according to Ley et al. (1970) and Gillis et al. (1970). ...
Article
The present study on Bacillus coagulans strain LBSC (DSM 17654) describes the use of whole genome sequencing, in correlation with the phenotypic properties to assess the safety of the strain. Analysis of the 16S rRNA sequence of the B. coagulans strain LBSC (DSM 17654), showed 100% homology with 99% coverage with B. coagulans strain HM-08. BLAT (BLAST Like Analysis Tool) analysis for whole genome comparison with B. coagulans ATCC 7050, B. coagulans HM-08 and B. coagulans Slac showed 96%, 99% and 99% sequence identity respectively. Whole genome sequencing results demonstrated a single scaffold of 36,35,902 bp and 3331 coding sequences. Gene ontology segregated the proteins as those with molecular function, cellular component and biological process of the predicted genes from assembled genome. Risk associated sequences like antibiotic resistance genes, biogenic amine producing genes, virulence factor genes and other safety related genes were identified with focus on horizontal gene transfer and its non-functionality. The absence of mobile elements in the vicinity of the genes, render it non-transferable and non-toxic phenotypic properties confirm the non-functionality of the genes. Absence of functional genes of concern and confirmation of absence of mobile elements in the vicinity of other non-clinically significant genes indicated no safety concern. The absence of complete and functional prophage sequences which are deleterious for the genome stability and presence of CRISPR system which are advantageous for genome stability by acting as a barrier to entry of foreign DNA elements indicated the stability of the genome. The molecular approach used in this study satisfies the requirements for the safety assessment of the probiotic strain which could indicate it to be potentially safe.
... DNA-DNA hybridization was performed to analyse the relationships of the novel isolate with related taxa based on the thermal denaturation principles and equations [29,30] and by following a previously described optimized procedure [31]. ...
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A Gram-stain-negative, short-rod, aerobic, non-motile, red to pink-pigmented bacterium, designated Fur1T, was isolated from the dry spikelet clusters of a plant called Setaria viridis near Dongguk University. Phylogenetic analysis conducted based on 16S rRNA gene sequences indicated that strain Fur1T belonged to the genus Hymenobacter of the family Hymenobacteraceae. The 16S rRNA gene of Fur1T showed highest sequence similarity to those of Hymenobacter metalli KACC 17381T (97.5 %) and Hymenobacter marinus KACC 19042T (97.1 %). Growth occurred at 4-37 °C (optimum, 25-28 °C), up to 1.0 % NaCl (optimum, 0 %) and pH 5.5-9.0 (optimum, pH 6.0-7.5). The major fatty acids of strain Fur1T were identified as iso-C15 : 0, C16 : 1 ω5c, anteiso-C15 : 0, summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 4 (comprising anteiso-C17 : 1B and/or iso-C17 : 1I) as the major cellular fatty acids. The predominant respiratory quinone was identified as MK-7. The polar lipids were phosphatidylethanolamine, five unidentified aminophospholipids, two unidentified phospholipids, one unidentified glycolipid and one unidentified polar lipid. The genomic DNA G+C content based on the draft genome sequence was 58.7 mol%. DNA-DNA relatedness between strain Fur1T and its closest relative was below 70 %. Characterization based on phylogenetic, chemotaxonomic and phenotypic analyses clearly indicated that strain Fur1T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter setariae sp. nov. is proposed. The type strain is Fur1T (=KACC 19903T=NBRC=113691T).
... DNA suspended in the 2X SSC and 1 : 10 000 SYBR Green was used for the analysis. DNA denaturation at 95.0 C was carried out followed by reassociation at optimum temperature 67.0 C. Fluorescence readings were taken every 10 s throughout the protocol and relative binding ratio was calculated according to De Ley et al. [20] and Gillis et al. [21]. ...
Article
A novel Gram-negative, rod shaped, non-motile bacterium, designated strain YK2T, was isolated from yak milk from Leh, India. The strain was positive for oxidase- and catalase-activities and negative for starch hydrolysis, nitrate reduction, citrate utilization, urease, lysine decarboxylase and ornithine decarboxylase activities. The predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c and/or iso-C15 : 0 2-OH (summed feature 3). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid and six unidentified lipids. The DNA G+C content of the strain was 38.9 mol%. The 16S rRNA gene sequence analysis indicated that strain YK2T was a member of the genus Sphingobacterium and closely related to Sphingobacterium alimentarium and Sphingobacterium composti with pair-wise sequence similarity of 98.3 and 97.9 %, respectively. The sequence similarity to other members of the genus Sphingobacterium was between 92.6 to 96.3 %. Phylogenetic analysis showed that strain YK2T clustered with Sphingobacterium alimentarium and together clustered with Sphingobacterium composti. DNA-DNA hybridization of strain YK2T with Sphingobacterium alimentarium WCC 4521T and Sphingobacterium composti T5-12T showed a relatedness of only 38 and 54 %, respectively. Based on the phenotypic characteristics and on phylogenetic inference, it appears that strain YK2T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium bovisgrunnientis sp. nov. is proposed. The type strain of Sphingobacterium bovisgrunnientis sp. nov. is YK2T (=MTCC 12631T=KCTC 52685T=JCM 31951T).
... DNA-DNA hybridization experiments were performed between strain MAH-6 T and closely related type strains of the genus Sphingomonas. The optimum renaturation temperature (44 °C) was calculated as [(0.51×G+C content)+47]−36 [24], where 36 °C is the correction for the presence of 50 % formamide [25]. Hybridization was performed with five replications for each sample. ...
Article
A novel bacterial strain, designated MAH-6T, was isolated from a garden soil sample. Cells were Gram-stain-negative, aerobic, non-motile and rod-shaped. The colonies were light yellow, smooth, circular and 0.6-1.2 mm in diameter when grown on nutrient agar for 3 days. Strain MAH-6T grew at 15-35 °C, at pH 5.0-7.0 and with 0-0.5 % NaCl. Cell growth occurred on nutrient agar and Reasoner's 2A (R2A) agar. The strain was positive for both catalase and oxidase tests. Cells were able to hydrolyse starch, aesculin, Tween 20 and Tween 80. According to 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Sphingomonas and was most closely related to Sphingomonas polyaromaticivorans B2-7T (98.2 % sequence similarity), Sphingomonas oligoaromativorans SY-6T (96.9 %) and Sphingomonas morindae NBD5T (96.6 %). The novel strain MAH-6T has a draft genome size of 4 370 740 bp (28 contigs), annotated with 4199 protein-coding genes, 46 tRNA and three rRNA genes. The genomic DNA G+C content of the strain was determined to be 66.2 mol% and the predominant isoprenoid quinone is Q-10. The major fatty acids were identified as summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c), C14 : 0 2OH and C16 : 0. The main polar lipids were phosphatidylcholine, sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on the results of phenotypic, genotypic, chemotaxonomic and DNA-DNA hybridization studies, strain MAH-6T represents a novel species, for which the name Sphingomonas chungangi sp. nov. is proposed, with MAH-6T as the type strain (=KACC 19292T=CGMCC1.13654T).
... The DNA of S. marcescens has the maximum GþC content among the enteric bacteria (Mandel and Rownd, 1964). The genome size of only one strain of S. marcescens has been determined (Gillis et al., 1970) as 3.57 Â 109 Da. The criterion of DNA relatedness clearly explains that the genus Serratia is different from all known genera of the family Enterobacteriaceae (Steigerwalt et al., 1976). ...
Chapter
Serratia sp. is a Gram-negative bacterium belongs to the family Enterobacteriaceae. More than 42 different species now have been associated with the genus Serratia of which almost ten species are presently recognized. Among them, five species viz, S. marcescens, S. plymuthicum, S. kilensis (sic), S. indica, and S. piscatorum were well characterized. Among them, S. plymuthica seem to be the most promising biocontrol agent. Serratia appears to be a omnipresent genus in nature. It is widely known as an insect pathogen and as a food-spoilage microorganism. Apart from that several strains of the genus exhibited plant growth promoting traits. Plant-associated Serratia consist of both endophytic and free-living species in the rhizosphere. Another remarkable characteristic trait of Serratia is the production of cell associated red color pigment, prodigiosin. The pigmented strains are non-pathogenic and used as biocontrol agent in agriculture. Prodigiosin has been studied to suppress growth of various bacteria, fungi, protozoans and also in viruses. Serratia also impart a significant role in phytoremediation by accumulating several toxic heavy metals from the soil. Thus the PGP strains of Serratia could be applied as a bio-inoculant for increased agricultural productivity and yield.
... DNA-DNA hybridization experiments were performed for strain MAH-12 T and its closely related reference strains. The optimum renaturation temperature (43 °C) was calculated as [(0.51 × G + C content) + 47] − 36 (Gillis et al. 1970), where 36 °C is the correction for the presence of 50% formamide (McConaughy et al. 1969). Hybridization was performed with five replications for each sample. ...
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A yellow-pigmented novel bacterial strain, MAH-12T, was isolated from a soil sample of Korean radish garden and was characterized using a polyphasic approach. Cells were Gram-staining negative, non-motile and rod-shaped. The strain was aerobic, catalase positive, optimum growth temperature and pH were 28–30 °C and 6.0, respectively. The novel strain is able to hydrolyze l-tyrosine, starch, esculin and 4-nitrophenyl-β-d-galactopyranoside. On the basis of 16S rRNA gene sequence analysis, strain MAH-12T belongs to the genus Sphingobium and is most closely related to several Sphingobium type strains (97.2–97.8%). In DNA–DNA hybridization tests, the DNA relatedness between strain MAH-12T and its closest phylogenetic neighbors was below 45.0%. The DNA G + C content was 64.0 mol% and the predominant respiratory quinone was ubiquinone-10. The major cellular fatty acids were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and C16:0. The DNA–DNA hybridization results and results of the genotypic analysis in combination with chemotaxonomic and physiological data demonstrated that strain MAH-12T represented a novel species within the genus Sphingobium, for which the name Sphingobium tyrosinilyticum is proposed. The type strain is MAH-12T (= KACC 19297T = CGMCC 1.16225T). The NCBI GenBank accession number for the 16S rRNA gene sequence of strain MAH-12T is KY964278 and the digital protologue database taxon number of strain MAH-12T is TA00463.
... DNA-DNA hybridization analysis was performed between strain S2T63 T and reference strains Microbacterium auranticum KACC 20510 T , Microbacterium kitamiens KACC 20514 T and Microbacterium laevaniformans KACC 14463 T . The re-association of DNA was carried out at the optimum re-association temperature of 73.0 C (with 10 % formamide) according to de Ley et al. [33], Gillis et al. [34] and Rossello-Mora et al. [35]. ...
Article
A cultivation-based study of the microbial diversity of cellular phone screens led to the isolation of a Gram-stain-positive, aerobic, rod-shaped and non-endospore-forming bacterium, designated S2T63T, exhibiting phenotypic and genotypic characteristics unique to the type strains of closely related species. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain is a member of Microbacterium, and most closely related to Microbacterium aurantiacum IFO 15234Tand Microbacterium kitamiense Kitami C2T. The DNA-DNA relatedness values of the strain S2T63Tto M. aurantiacum KACC 20510T, M. kitamiense KACC 20514Tand Microbacterium laevaniformans KACC 14463Twere 65 % (±4), 29.5 % (±3) and 55.9 % (±4), respectively. The genomic DNA G+C content was 71.8 mol%. The major fatty acids were anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and anteiso-C17 : 0. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unidentified polar lipids. The peptidoglycan contained the amino acids glycine, lysine, alanine and glutamic acid, with substantial amounts of hydroxy glutamic acid detected, which is characteristic of peptidoglycan type B1α. The predominant menaquinones were MK-12 and MK-13. Rhamnose, fucose and galactose were the whole-cell sugars detected. The strain also showed biofilm production, estimated by using crystal violet assay. Based on the results of the phenotypic and genotypic characterizations, it was concluded that the new strain represents a novel species of the genus Microbacterium, for which the name Microbacteriumtelephonicum is proposed, with S2T63T (=MCC 2967T=KACC 18715T=LMG 29293T) as the type strain.
... To assess the DNA G+C content and DNA-DNA hybridization, total genomic DNA was extracted and purified following the method of Ausubel et al. [22]. The DNA G+C content of strain S8 T was determined by following a fluorimetric method [23] performed to assess relatedness of the novel isolate with related taxa on the basis of the denaturation principles and equations of De Ley et al. [24] and Gillis et al. [25] and an optimized protocol described by Loveland-Curtze et al. [26]. The extent of DNA-DNA hybridization was determined using the fluorimetric method with real-time PCR. ...
Article
A novel Gram-stain-negative bacterium, designated strain S8T, was isolated from a soil sample obtained in Gyeonggi Province, Republic of Korea. Cells of strain S8Twere endospore-forming, motile by means of peritrichous flagella, and rod-shaped. S8Tcolonies were round, convex, wavy and white. Strain S8Tgrew optimally at 37 °C, pH 6-8, and up to 2.0 % (w/v) NaCl. Based on 16S rRNA gene sequence similarity, strain S8Twas affiliated with the genus Paenibacillus in the family Paenibacillaceae and was most closely related to Paenibacillus yonginensis DCY84Tand Paenibacillus physcomitrellae XBT(98.8 and 97.1 % sequence similarity). The DNA G+C content of the novel strain was 53.1±0.3 mol%. Strain S8Tcontained diphosphatidylglycerol, phosphatidylglycerol, two phospholipids, four aminophospholipids, an aminolipid and three unidentified lipids. The major fatty acid was anteiso-branched C15 : 0. The quinone was menaquinone MK-7. The peptidoglycan of strain S8Tcontained meso-diaminopimelic acid. The DNA-DNA hybridization values of strain S8Twith P. yonginensis KCTC 33428Tand P. physcomitrellae DSM 29851Twere 44 % and 32 %, respectively. Data from the DNA-DNA hybridization, biochemical, phylogenetic and physiological analyses indicate that strain S8T(=KCTC 33848T=JCM 31672T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillusmobilis sp. nov. is proposed.
... The resulted DNA was mixed with SYBR Green-I (Invitrogen), a fluorescent dye specifically binds to double stranded DNA (dsDNA). The relative rate of reassociation of homoduplex and heteroduplex DNA was analysed by denaturation followed by optimum reassociation (Gillis et al. 1970) by using StepOnePlus Real-Time PCR system (Applied Biosystems). DNA-DNA relatedness in terms of relative binding ratio was calculated as described elsewhere (De Ley et al. 1970). ...
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Two Gram-stain positive, endospore forming, non-motile, rod shaped bacterial strains SN6T and SN6b were isolated from scats of a mildly venomous vine snake (Ahaetulla nasuta). Strains were phenotypically resistant to multiple antibiotics of four different classes i.e. aminoglycosides, β-lactams, fluoroquinolones and sulphonamides. Cells of both the strains were catalase positive and oxidase negative. Phylogenetic analysis based on 16S rRNA gene sequence analysis of these two strains showed closest similarity (99.2% and 99.3%) with Savagea faecisuis Con12T, the only species of the genus Savagea and ≤ 94.9% with the species of other closest genera of the family Planococcaceae. The 16S rRNA gene sequence similarity (99%), DNA-DNA relatedness (95%) and similar phenotypic characteristics between the strains SN6T and SN6b revealed their phylogenetic affiliation to the same species. Hence, strain SN6b is an additional strain of the type strain SN6T. DNA-DNA relatedness of strain SN6T with S. faecisuis Con12T was 32.8%. Predominant fatty acids were iso-C15:0 (32.0%), iso-C16:1 ω11c (19.2%) and iso-C17:1 ω10c (12.1%). MK-6 (100%) was the only respiratory quinone of strain SN6T. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. Cell wall peptidoglycan was A4α; L-Lys-Gly-D-Glu type. The DNA G + C content (mol%) of SN6T was 40.8. Whole genome sequence of SN6T consisted of 26,37,389 base pairs in length with 2667 annotated genes, out of which 1021 corresponds to hypothetical proteins and 1646 with functional assignments including antibiotic resistance, multidrug resistance efflux pumps, invasion and virulence factors. Comparative polyphasic study of the strains SN6T, SN6b and S. faecisuis Con12T elucidated the differentiating characteristics which led to describing strain SN6T and SN6b as a novel species of the genus Savagea for which the name Savagea serpentis sp. nov is proposed. The type strain of Savagea serpentis is SN6T (= KCTC 33546T = CCUG 6786T).
... The DNA G?C content of strain AL-54 T was determined by reversed-phase HPLC using the method of Mesbah et al. (1989) with Escherichia coli DH5a (CICC 10399) as a control. DNA-DNA hybridization (DDH) experiments were carried out to evaluate the DDH relatedness between strain AL-54 T and its reference strains using the optical renaturation rate method (Gillis et al. 1970) and a Perkin Elmer Lambda 35 UV/VIS spectrophotometer by the service of CICC (for P. chengduensis CGMCC 2318 T by the service of CGMCC). ...
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A Gram-stain negative, aerobic, rod-shaped, motile by a single polar flagellum, non-spore-forming bacterium, designated strain AL-54T, was isolated from the storage liquid in the stems of Populus euphratica tree at the ancient Ugan River in Xinjiang, PR China. Isolated AL-54T grew optimally at pH 7.0 and temperature 35 °C in the presence of 3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that the isolate belonged to the genus Pseudomonas and was closely related to Pseudomonas songnenensis NEAU-ST5-5 T (97.6%), Pseudomonas zhaodongensis NEAU-ST5-21 T (97.5%), Pseudomonas alcaliphila AL15-21T (97.3%), Pseudomonas toyotomiensis HT-3T (97.3%), Pseudomonas oleovorans subsp. lubricantis RS1T (97.3%), Pseudomonas stutzeri ATCC 17588T (97.3%), Pseudomonas chengduensis CGMCC 2318T (97.2%), and Pseudomonas xanthomarina KMM 1447T (97.1%). Multilocus Sequences Analysis (MLSA) of strain AL-54T based on the three housekeeping genes, rpoB, rpoD and gyrB further confirmed the phylogenetic assignment of the isolates. The G+C content was 64.7 mol%. The DNA-DNA hybridization with P. songnenensis NEAU-ST5-5 T, P. zhaodongensis NEAU-ST5-21T, P. alcaliphila AL15-21T, P. toyotomiensis HT-3T, P. oleovorans subsp. lubricantis RS1T, P. stutzeri ATCC 17588T, P. chengduensis CGMCC 2318T and P. xanthomarina KMM 1447T revealed 44.0%, 44.7%, 60.1%, 48.7%, 49.1%, 60.1%, 58.9% and 60.2% relatedness respectively. The predominant quinone system is ubiquinone-9 (Q-9). The major components of the cellular fatty acids (>10%) were summed feature 8 (comprising C18:1 ω7c /C18:1 ω6c), summed feature 3 (comprising C16:1 ω7c /C16:1 ω6c) and C16:0. The detected major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (PC). On the basis of phenotypic data, chemotaxonomic and phylogenetic properties, strain AL-54T can consider as a novel species within the genus Pseudomonas, for which the name Pseudomonas lopnurensis sp. nov. is proposed. The type strain is AL-54T (= JCM 19136T = CCTCC AB 2013066T = NRRL B-59987T).
... The chromosomal material seems to consist of two classes of DNA of unique sequence. This conclusion was suggested by studies of initial renaturation rates (52) was of the highest G+C content. Since plasmids have not been detected in lysates of cells analyzed in CsCl-ethidium bromide gradients (Sadoff, unpublished data), these two types of DNA must be constituents of the A. vinelandii chromosome. ...
... The genome size of some 40 Zymomonas strains was determined with the initial renaturation rate method (68,69 Genome-DNA Relatedness We determined the degree of genome-DNA relatedness (percent D; DNA "homology") (163) with the initial renaturation rate method (48) in stringent conditions, i.e., those that allow duplex formation to occur only between very closely related or identical polynucleotide sequences. ...
... An aliquot of DNA suspended in 2Â SSC buffer was used to analyse the reassociation of homoduplex and heteroduplex DNA, which was carried out at an optimum renaturation temperature of 70.6 C [T or =0.51Â(% G+C)+47.0] [15] based on the protocol along with 10 % formamide. The experiment was performed in triplicate. ...
Article
A Gram-stain-negative, yellowish-orange pigmented, rod-shaped, motile bacterium, designated strain ARC111T, was isolated from sediment of Arctic permafrost at Midtre Lovénbreen glacier, Svalbard. 16S rRNA gene based identification of strain ARC111Tdemonstrated highest sequence similarities to Subsaxibacter broadyi P7T(97.8 %) and Subsaxibacter arcticus JCM30334T(97.5 %) and ≤95.2 % with all other members of the family Flavobacteriaceae. Phylogenetic analysis revealed the distinct positioning of strain ARC111Twithin the genus Subsaxibacter. The G+C content of ARC111Twas 37.8±0.5 mol% while DNA-DNA hybridization depicted 35.6 % relatedness with S. arcticus JCM30334T. Strain ARC111Thad C15 : 0iso, C16 : 0iso 3-OH, C15 : 1iso G, C15 : 0anteiso, C16 : 1iso H and C17 : 0iso 3-OH as major (>5 % of the total) cellular fatty acids and MK-6 was the predominant respiratory quinone. The polar lipid profile of strain ARC111Tconsisted of phosphatidylethanolamine, aminolipid and an unidentified lipid. Strain ARC111Tharboured sym-homospermidine as the major polyamine. Characteristic differences obtained using polyphasic analysis of strain ARC111Tand its closest relatives suggested that strain ARC111Tis a novel species of genus Subsaxibacter, for which the name Subsaxibacter sediminis sp. nov. has been proposed. The type strain is ARC111T(=MCC 3191T=KCTC 42965T=LMG 29783T=GDMCC 1.1201T).
... The genome sizes of all strains examined were in the range of 2,200 x 106 to 3,000 x 106 daltons. This range is typical for species of the Enterobacteriaceae (2,12,14). In general, K. pneumoniae strains with genome sizes similar to 13883, showed the highest relative reassociation values to this human strain. ...
Article
The phenotypic and nucleic acid properties of Klebsiella pneumoniae have been studied on cultures obtained from six different habitats (humans, vegetables, seeds, trees, rivers, and pulp mills). The 19 cultural reactions of 107 isolates varied significantly only in tryptophanase activity and dulcitol fermentation. The percentage of guanine plus cytosine base composition of 41 isolates varied from 53.9 to 59.2%. The range of percentage of guanine plus cytosine base composition for environmental klebsiellas was broader than that for the cultures of human origin. The range of deoxyribonucleic acid relative reassociation (homology) to the human K. pneumoniae reference strain extended from 5% to 100% and the chromosome molecular weights ranged from 2,200 × 10⁶ to 3,000 × 10⁶. The species of K. pneumoniae is thus molecularly more heterogeneous than previously thought and most isolates of human, pulp mill, and river origin are genetically indistinguishable. The presence of K. pneumoniae therefore represents a deterioration of the microbiological quality of the environment and should be considered of public health significance. At the present time the health significance of the molecularly more divergent strains, primarily of vegetable and seed origin, their relationship to klebsiellas of human origin, or to other genera of the Enterobacteriaceae is unclear.
... t = 0.06) of the SP/3 prophage (222). These calculations suggest that SP,8 ( [11,56] ...
... DNA-DNA hybridization was also performed with the strain with the closest similarities in biochemical and physiological characteristics and 16S rRNA gene sequences. The levels of DNA-DNA hybridization were determined using a modified optical renaturation method described by De et al. (1970) and Gillis et al. (1970). ...
Article
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Biological control agents (BCAs) are considered as one of the most important strategies for controlling Fusarium wilt, and bioorganic fertilizer, in particular, has been extensively investigated. However, little is known regarding how a biocontrol microorganism affects the suppression mechanisms when combined with different amendments. In this study, a pot experiment was performed using banana plants to investigate the different mechanisms by which the biocontrol bacterium Bacillus velezensis HN03 (isolated from our laboratory) and amendments suppress Fusarium wilt. The incidence of banana wilt was decreased under HN03 and was reduced further when HN03 was combined with compost, particularly wormcast. In the suppression of Fusarium wilt, HN03 was found to influence the soil environment in various ways. HN03 increased the peroxidase level, which improves plant defense, and was highest when combined with wormcast, being 69 times higher than when combined with cow dung compost. The high accumulation of Mg and P in the “HN03 + wormcast” and Zn and Mn in the “HN03 + cow dung” treatments was negatively correlated with disease incidence. Furthermore, HN03 re-established the microbial community destroyed by the pathogen and further increased the level of suppression in the wormcast. HN03 also enhanced the functional traits of the soil, including defensive mechanism-related traits, and these traits were further enhanced by the combination of HN03 + wormcast.
... This experiment was done between strain MAH-20 T and two closely related type strains of genus Sphingomonas. The optimum renaturation temperature (45 °C) was calculated as [(0.51 × G + C content) + 47] − 36 (Gillis et al. 1970), where 36 °C is the correction for the presence of 50% formamide (McConaughy et al. 1969). Five replications were used for each sample. ...
Article
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A novel bacterial strain, designated MAH-20T, was isolated from a soil sample of a tomato garden. Cells of strain MAH-20T were Gram-stain negative, aerobic, motile, and rod-shaped. The colonies were light brown colored, smooth, spherical, and 0.2-0.7 mm in diameter when grown on Luria-Bertani agar for 2 days. Strain MAH-20T grows at 15-40 °C (optimum growth temperature 30-32 °C), at pH 5.0-10.0 (optimum growth pH 7.0) and at 0-2.0% NaCl. The strain showed positive activity for both oxidase and catalase tests. Cells were able to hydrolyze starch, DNA, urea, gelatin, L-arginine, and Tween 20. According to the 16S rRNA gene sequence similarity, the strain MAH-20T was identified as a new member of the genus Sphingomonas and had the close sequence similarity with Sphingomonas changbaiensis V2M44T (98.9%) and Sphingomonas tabacisoli X1-8T (98.1%). The genomic ANI value between strain MAH-20T and S. changbaiensis NBRC 104936T was 84.4%. The novel strain MAH-20T has a draft genome size of 3,350,026 bp (25 contigs), annotated with 3210 protein-coding genes, 46 tRNA, and 3 rRNA genes. The genomic DNA G + C content of isolate was 67.3 mol%, the predominant quinone was ubiquinone 10 and the major fatty acids were C16:0, C17:1 ω6c and summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c). On the basis of DNA-DNA hybridization results, phenotypic, genotypic, and chemotaxonomic data, the isolated strain MAH-20T represents a novel species, for which the name Sphingomonas horti sp. nov. is proposed, with MAH-20T as the type strain (= KACC 19746T = CGMCC1.13658T).
... All six qPCRs targeting these Salmonella loci yielded highly satisfactory efficiency and linearity over a broad template range based upon representative runs performed during the course of this study (Table 3). Using 10-fold serial dilutions of Salmonella genomic DNA, all six qPCRs consistently detected their respective DNA targets ranging in amount from 100 ng down to 100 fg, a DNA amount corresponding to 20 CFUs of Salmonella (Gillis et al. 1970), with inconsistent detection of 10 fg of genomic DNA (2 CFUs). Media spiked with known titers of Salmonella Enteritidis (isolate No. 44) helped establish an optimal DNA extraction protocol for the three different media types and determine the amount of pCR2.1TopoinvAHAV5 ...
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A real-time PCR (qPCR) regimen, using up to six genetic targets, was developed to rapidly detect Salmonella and in particular identify Salmonella Enteritidis. The test regimen was first evaluated using a reference culture collection of Salmonella to confirm the appropriateness of the selected targets, which included up to three genetic markers for discrimination of Salmonella Enteritidis from other Salmonella serovars commonly found in poultry facilities. The qPCR procedure was then compared with culture methods used to detect Salmonella using a collection of enrichment broths previously generated from 239 environmental samples collected from a large number of hatchery facilities across Canada over several years. The qPCR regimen facilitated specific detection of Salmonella Enteritidis, and on a sample basis, it showed excellent agreement with the culture methods. Moreover, in many cases, qPCR detected Salmonella earlier in the culture process than did the culture method. Application of this method will significantly shorten test times and allow more timely identification of infected poultry premises, thereby improving present programmes aimed at controlling Salmonella Enteritidis at the environmental source.
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A yellow pigmented, Gram-staining negative, motile and rod-shaped novel bacterial strain, designated MAH-14T was isolated from rhizospheric soil and was characterized using a polyphasic approach. The isolated strain was aerobic, oxidase and catalase were positive, optimum growth temperature and pH were 28–30 °C and 6.5, respectively. The novel strain is able to hydrolyze casein, starch, esculin, gelatin, l-tyrosine, DNA, tween 80, tween 20, l-arginine and 4-nitrophenyl-BD-galactopyranoside. On the basis of 16S rRNA gene sequence analysis, strain MAH-14T belongs to the genus Luteibacter and is most closely related to Luteibacter yeojuensis R2A16-10T (98.5%), Luteibacter anthropi CCUG 25036T (98.4%) and Luteibacter rhizovicinus LJ96T (98.3%). In DNA–DNA hybridization experiments, the DNA relatedness between strain MAH-14T and its closest phylogenetic neighbor was below 45.0%. The predominant respiratory quinone and the DNA G + C content of the novel strain were ubiquinone-8 and 63.5 mol%, respectively. The novel strain MAH-14T is able to produce flexirubin-type pigments. The major cellular fatty acids were C15:0 iso, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 9 (C17:1 iso ω9c and/or C16:0 10-methyl). The DNA–DNA hybridization results and results of the genotypic analysis in combination with chemotaxonomic and physiological data revealed that strain MAH-14T represented a novel species within the genus Luteibacter, for which the name Luteibacter pinisoli, is proposed. The type strain is MAH-14T (= KACC 19298T = CGMCC 1.16227T).
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The taxonomic position of a Gram-stain-negative, rod-shaped bacterial strain, designated PI11T, isolated from the rhizospheric sediment of Phragmites karka was characterized using a polyphasic approach. Strain PI11T could grow optimally at 1.0% NaCl concentration with pH 7.0 at 30°C and was positive for oxidase and catalase but negative for hydrolysis of starch, casein, and esculin ferric citrate. Phylogenetic analysis of 16S rRNA gene sequences indicated that the strain PI11T belonged to the genus Pseudomonas sharing the highest sequence similarities with Pseudomonas indoloxydans JCM 14246T (99.72%), followed by, Pseudomonas oleovorans subsp. oleovorans DSM 1045T (99.29%), Pseudomonas toyotomiensis JCM 15604T (99.15%), Pseudomonas chengduensis DSM 26382T (99.08%), Pseudomonas oleovorans subsp. lubricantis DSM 21016T (99.08%), and Pseudomonas alcaliphila JCM 10630T (99.01%). Experimental DNA-DNA relatedness between strain PI11T and P. indoloxydans JCM 14246T was 49.4%. The draft genome of strain PI11T consisted of 4,884,839 bp. Average nucleotide identity between the genome of strain PI11T and other closely related type strains ranged between 77.25–90.74%. The polar lipid pattern comprised of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine. The major (> 10%) cellular fatty acids were C18:1ω6c/ω7c, C16:1ω6c/ω7c, and C16:0. The DNA G + C content of strain PI11T was 62.4 mol%. Based on the results of polyphasic analysis, strain PI11T was delineated from other closely related type strains. It is proposed that strain PI11T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas sediminis sp. nov. is proposed. The type strain is PI11T (= KCTC 42576T = DSMZ 100245T).
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A novel bacterial strain MAH-10Twas isolated from soil sample of a Chinese cabbage garden, Republic of Korea and was characterized using a polyphasic approach. Cells were Gram-staining negative, rod-shaped, yellowish orange colored, and motile. The strain was aerobic, catalase and oxidase are positive, and optimum growth temperature and pH were 28 °C and 6.5, respectively. Flexirubin-type pigments were found to be present. On the basis of 16S rRNA gene sequence analysis, strain MAH-10Tbelongs to the genus Flavobacterium and is most closely related to Flavobacterium tyrosinilyticum KCTC 42726T(98.7%). On the basis of phylogenetic tree, other closely related species are Flavobacterium banpakuense KACC 14225T(98.3%) and Flavobacterium chungbukense KACC 15048T(97.6%). In DNA-DNA hybridization tests, the DNA relatedness between strain MAH-10Tand its closest phylogenetic neighbor was below 45.0%. The DNA G+C content was 37.2 mol% and the predominant respiratory quinone was menaquinone-6. The major cellular fatty acids were C15:0iso, C16:0, and summed feature 3 (C16:1ω7c and/or C16:1ω6c). On the basis of DNA-DNA hybridization results and genotypic, chemotaxonomic, and physiological data analysis, it is demonstrated that strain MAH-10Trepresented a novel species within the genus Flavobacterium, for which the name Flavobacterium chungangensis is proposed. The type strain is MAH-10T(=KACC 19296T=CGMCC 1.16226T). The NCBI GenBank accession number for the 16S rRNA gene sequence of strain MAH-10Tis KY964277 and the digital protologue database (DPD) Taxon Number of strain MAH-10Tis TA00296.
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The replication of R124, and a copy mutant derivative of it, was measured with respect to dependence on the host DnaA, DnaB, DnaC, DnaE, DnaG, and Po1A gene products. Both plasmids replicated under conditions where the DnaA gene product was inactivated or where the polymerising activity of the PolA gene product was reduced. In contrast, neither plasmid replicated to any appreciable extent, if the DnaB, DnaC, DnaE or DnaG gene products were inactivated. R124 integratively suppressed the lesion of the dnaA mutant but the copy mutant derivative had only a very weak suppressing effect. Neither plasmid suppressed the lesions of any of the other dna mutants.
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A Gram-stain-negative, rod-shaped bacterial strain designated as 20VBR1 T was isolated from a valley glacier (Vestrebroggerbreen) snout ice sample from Ny-Ålesund, Svalbard, Arctic. The colonies were smooth, circular and light creamish on half-strength R2A agar and grew at 10–35 °C (optimum, 20 °C), at pH 6.5–8.0 (optimum, 7.0) and with 0–2.5 % (w/v) NaCl (optimum, 0.5 %). 16S rRNA gene sequence analysis revealed that strain 20VBR1 T belonged to the genus Phenylobacterium and was most closely affiliated to Phenylobacterium aquaticum W2-3-4 T (97.65 % similarity), Phenylobacterium haematophilum LMG 11050 T (97.57 %) and Phenylobacterium koreense Slu-01 T (96.91 %). 20VBR1 T has a genome size of 4.24 Mb, comprising 4185 predicted genes with a DNA G+C content of 67.86 mol%. DNA–DNA hybridization experiments indicated that the DNA–DNA relatedness between strain 20VBR1 T and P. aquaticum KACC 18306 T was 41.95±4.36 %, well below the threshold (<70 %) to delineate bacterial species. Genome relatedness indexes revealed that the average nucleotide identity and digital DNA–DNA hybridization values between 20VBR1 T and its closest phylogenomic relative, P. aquaticum KACC 18306 T , were 78.97 and 22.10 %, respectively. The predominant isoprenoid quinone was ubiquinone (Q-10) and the major polar lipids were phosphatidylglycerol, one unknown phospholipid, one unknown glycolipid and four unidentified polar lipids. The major fatty acids (>10 %) of strain 20VBR1 T were summed feature 8 (comprising C 18 : 1 ω 7 c and/or C 18 : 1 ω 6c), summed feature 3 (comprising C 16 : 1 ω 6 c and/or C 16 : 1 ω 7 c ) and C 16 : 0 . Based on the physiological, biochemical, chemotaxonomic, phylogenetic and phylogenomic analyses, isolate 20VBR1 T is considered to represent a novel species of the genus Phenylobacterium , for which the name Phenylobacterium glaciei sp. nov. is proposed. The type strain is 20VBR1 T (=JCM 33227 T =DSM 111428 T =MCC 4220 T ).
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A strictly aerobic, haloalkali-tolerant, Gram-stain-positive, non-motile, rod-shaped bacterium, designated strain SMB4T, was isolated from a water sample collected from Sambhar salt lake, Rajasthan, India. Growth occurred at 25-50 °C, 4-12 % (w/v) NaCl and pH of 5-9. Strain SMB4T was positive for β-galactosidase, oxidase, catalase and urease activities. The fatty acids were dominated by branched forms of fatty acids with iso- and anteiso-saturated fatty acids, with a high abundance of anteiso-C15 : 0, anteiso-C17 : 0 and C18 : 0. The cell-wall peptidoglycan of strain SMB4T contained meso-diaminopimelic acid, while the polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and three unidentified lipids. The DNA G+C content of strain SMB4T was 49.1 mol%. A blast sequence similarity search based on 16S rRNA gene sequence indicated that Salibacterium halochares, Salibacterium halotolerans and Salibacterium qingdaonense were the nearest phylogenetic neighbours, with a pair-wise sequence similarities of 98.4, 98.2 and 97.0 % respectively. Phylogenetic analysis showed that strain SMB4T was clustered with S. halochares and together clustered with S. halotolerans and S. qingdaonense. DNA-DNA hybridization of strain SMB4T with S. halochares DSM 21373T, S. halotolerans S7T and S. quigdaonense DSM 21621T showed a relatedness values of only 39.8, 26.3 and 42.8 %, respectively. Based on its phenotypic characteristics and on phylogenetic inference, strain SMB4T represents a novel species of the genus Salibacterium, for which the name Salibacterium nitratireducens sp. nov. is proposed. The type strain is SMB4T (=MTCC 12633T=KCTC 33876T=JCM 32187T).
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Assessment of the bacterial diversity associated with a decaying fern, Athyrium wallichianum Ching, revealed the presence of a novel bacterial strain named M46T. It was Gram-stain-negative, rod-shaped, non-motile and aerobic with cellulose and xylan degradation abilities. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain M46T was affiliated to the genus Sphingobacterium, exhibiting the highest sequence similarity of 97.9 % to Sphingobacterium ginsenosidimutans THG 07T, Sphingobacterium canadense CR11T and Sphingobacterium detergens6.2 ST. Multilocus sequence analysis (MLSA) based on concatenated sequences of the rpoB, cpn60 and 16S rRNA genes showed that strain M46T clustered together with S. canadense CR11T. The genome of strain M46T had a G+C content of 40.6 mol% and chromosome of 6 853 865 bp. Average nucleotide identity (ANI) between strain M46T and S. detergens 6.2 ST and S. siyangense SY1T was 85.1 and 78.1 %, respectively. DNA-DNA relatedness values among strain M46T and other closely related Sphingobacterium species were <70 %. ANI and DNA-DNA relatedness findings strongly supported M46T as a putative novel strain of Sphingobacterium. The predominant fatty acids of strain M46T were iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and iso-C17 : 0 3-OH, and MK-7 was the dominant isoprenoid quinone. The polar lipid profile of strain M46T contained phosphatidylethanolamine as the dominant component, while minor amounts of phosphoglycolipid, one unidentified aminophospholipid, two unidentified phospholipids and four unidentified lipids were also detected. Based on 16S rRNA gene sequence similarities, MLSA results, genomic characteristics, and phenotypic and biochemotaxonomic analyses, strain M46T is considered to represent a novel species in the genus Sphingobacterium, for which the name Sphingobacterium athyrii sp. nov. is proposed. The type strain is M46T (=CGMCC 1.13466T=JCM 32543T).
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Antibiotic-resistance is ever growing burden on our society for the past many years. Many synthetic chemistry approaches and rational drug-design have been unable to pace up and tackle this problem. Natural resources, more specifically, the microbial diversity, on the other hand, make a traditional and still the best platform to search for new chemical scaffolds and compounds. Here, we report the antimicrobial characteristics of novel bacterial isolate from a salt lake in India. We screened the bacterial isolates for their inhibitory activity against indicator bacteria and found that four novel species were able to prevent the growth of test strains studied in vitro. Further, we characterized one novel species (SMB1T = SL4-2) using polyphasic taxonomic approaches and also purified the active ingredient from this bacterium. We successfully characterized the antimicrobial compound using mass spectroscopy and amino acid analysis. We also allocated two novel biosynthetic gene clusters for putative bacteriocins and one novel non-ribosomal peptide gene cluster in its whole genome. We concluded that the strain SMB1T belonged to the genus Paenibacilllus with the pairwise sequence similarity of 98.67% with Paenibacillus tarimensis DSM 19409T and we proposed the name Paenibacillus sambharensis sp. nov. The type strain is SMB1T (=MTCC 12884 = KCTC 33895T).
Chapter
When compared to other bacteria, the genus Streptomyces involves a distinct assembly of Gram-positive bacteria that are universal in nature and display complex range in colony color, secretion of pigments, etc. The greatest role of Streptomyces is its capacity to generate biologically active specialized products, for instance, antivirals, antihypertensives, anticancer, fungicides, immunosuppressors, and most importantly antibiotics. Taxonomy or systematics of bacteria involves the study of a range of organisms that makes use of classification, nomenclature, and identification. Streptomyces taxonomy is established on limited characters such as structural coloration, bionomical necessities, and structural aspect of chains of fungal spore morphology. Streptomyces have been classified based on different approaches like the International Streptomyces Project (ISP) of 1966, genomic sequence (phylogenetic approach), molecular techniques (genotyping approach), chemical characteristics (chemotaxonomy), or numerical taxonomy. As far as the diversity is concerned, in the majority of the bacterial domain, the genus Streptomyces is highly diversified and is scattered around substantial terrestrial sections.
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A systematic examination of a variety of isolates of the bacterial endoparasite Bdellovibrio has revealed extensive molecular diversity. The quantity of deoxyribonucleic acid (DNA) polynucleotide homology ranges from more than 90% among the isolates with DNA containing 50 to 51% guanine plus cytosine (GC) to undetectable levels between the 43% GC and 51% GC isolates. The two isolates with low GC-containing DNA (H-I Bdellovibrio A3.12 and UKi2) have only 16% DNA homology. H-I Bdellovibrio A3.12 and 109 have barely detectable ribosomal ribonucleic acid (rRNA) homology, whereas the homology approaches 100% among all the high GC isolates tested. Cases of high DNA/DNA and DNA/rRNA homologies are reflected in low dissimilarities of enzyme migration patterns in starch gel electrophoresis. The dissimilarities exhibited among the high GC Bdellovibrio isolates are as low as those previously reported for different Escherichia coli strains. The zymograms of H-I Bdellovibrio A3.12 and UKi2 are completely different from each other as well as from all other bdellovibrios (100% dissimilarity). Genome sizes determined for the representative isolates demonstrate three size ranges which coincide with group differences based on the above measurements. Enzyme assays reveal that all isolates possess a tricarboxylic acid cycle and most contain an alanine and glutamic dehydrogenase. We conclude that the use of bacterial endoparasitism as a defining trait has resulted in a molecularly diverse collection of isolates. It is recommended that the specific epitaph bacteriovorus be used only for the type specimen (Bdellovibrio 100 of Stolp and Starr, 1963) and for other related 50 to 51% GC isolates. The heterogeneity of the group warrants two new species. We designate Bdellovibrio A3.12 as the nomenclatural type of B. starrii sp. n. and Bdellovibrio UKi2 as the nomenclatural type of B. stolpii sp. n.
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A novel nocardioform strain, CICC 11023T, was isolated from a tissue biopsy of neck lesions of a patient with primary cutaneous nocardiosis and characterized to establish its taxonomic position. The morphological, biochemical, physiological and chemotaxonomic properties of strain CICC 11023T were consistent with classification in the genus Nocardia. Whole-cell hydrolysates were rich in meso-diaminopimelic acid, galactose, arabinose and fructose. Mycolic acids were present. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and two unidentified lipids, and the predominant menaquinone was cyclo MK-8 (H4, ω-cyclo). The main fatty acids (>5 %) were C18 : 0 10-methyl (TBSA), C16 : 0, summed feature 4 (C16 : 1 trans 9/C15 : 0 iso 2OH), C15 : 0 and C17 : 0 10-methyl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the isolate is most closely related (>98 % similarity) to the type strains Nocardia ninae OFN 02.72T, Nocardia iowensis UI 122540T and Nocardia alba YIM 30243T, and phylogenetic analysis of gyrB gene sequences showed similarity (89.1-92.2 %) to Nocardia vulneris NBRC 108936T, Nocardia brasiliensis IFM 0236T and Nocardia exalbida IFM 0803T. DNA-DNA hybridization results for strain CICC 11023T compared to Nocardia type strains ranged from 20.4 to 35.4 %. The genome of strain CICC 11023T was 8.78 Mbp with a G+C content of 67.4 mol% overall. The average nucleotide identity (ANI) values between strain CICC 11023T and N. alba YIM 30243T were low (OrthoANIu=77.47 %), and the ANI values between strain CICC 11023T and N. vulneris NBRC 108936 T were low (OrthoANIu=83.75 %). Consequently, strain CICC 11023T represents a novel Nocardia species on the basis of this polyphasic study, for which the name Nocardia colli sp. nov. is proposed. The type strain is CICC 11023T (=KCTC 39837T).
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A Gram-stain-negative, aerobic, non-motile and rod- or coccoid-shaped novel bacterial strain, designated MAH-25T, was isolated from soil sampled in a pine garden. The colonies were observed to be light pink-coloured, smooth, spherical and 1-2 mm in diameter when grown on nutrient agar for 2 days. Strain MAH-25T was found to be able to grow at 15-35 °C, at pH 5.0-8.0 and at 0-2.0 % NaCl. Cell growth occurred on Reasoner's 2A agar and nutrient agar. The strain was found to be positive in both oxidase and catalase tests. According to 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Ramlibacter and closely related to Ramlibacter solisilvae 5-10T (98.0 % similarity), Ramlibacter henchirensis TMB834T (97.7 %), Ramlibacter tataouinensis TTB310T (97.6 %) and Ramlibacter rhizophilus YS3.2.7T (97.3 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain MAH-25T and the four closely related type strains were in the range of 78.8-81.3 % and 22.3-24.1 %, respectively. The novel strain MAH-25T has a draft genome size of 5 505 957 bp (11 contigs), annotated with 5210 protein-coding genes, 46 tRNA and three rRNA genes. The genomic DNA G+C content was determined to be 70.3 mol%. The predominant isoprenoid quinone was ubiquinone 8 (Q-8). The major fatty acids were identified as C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of DNA-DNA hybridization, genotypic analysis, chemotaxonomic and physiological data, strain MAH-25T represents a novel species within the genus Ramlibacter, for which the name Ramlibacter pinisoli sp. nov. is proposed, with MAH-25T (=KACC 19839T=CGMCC1.13660T) as the type strain.
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The genome sizes of Streptomyces coelicolor and Streptomyces rimosus as calculated by deoxyribonucleic acid reassociation kinetics are approximately 10.5 × 10⁶ nucleotide pairs.
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Genetic relatedness among Staphylococcus aureus typing bacteriophages 80, 47, 81, 71, 77, and 187 was investigated by using base ratio determinations and deoxyribonucleic acid (DNA)-DNA hybridization. Guanine/cytosine (G/C) content, as determined by thermal denaturation and chromatographic analysis of the purines released by acid hydrolysis of the DNA, was between 31 and 36%. No pattern correlating G/C content with serological or lytic group was discernible. DNA-DNA hybridization studies indicated high degrees of homology (43% or more) among the genomes of phages in the same serological group. Less homology (29% or less) was observed between the genomes of phages belonging to different serological groups. These findings implied a positive correlation between serological and genetic relatedness.
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OrganiCam is a laser-induced luminescence imager and spectrometer designed for standoff organic and biosignature detection on planetary bodies. OrganiCam uses a diffused laser beam (12° cone) to cover a large area at several meters distance and records luminescence on half of its intensified detector. The diffuser can be removed to record Raman and fluorescence spectra from a small spot from 2 m standoff distance. OrganiCam's small size and light weight makes it ideal for surveying organics on planetary surfaces. We have designed and built a brassboard version of the OrganiCam instrument and performed initial tests of the system.
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Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid.
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Two bacterial strains, designated MJB4 T and SJ7 T , were isolated from water samples collected from Jeongbang Falls on Jeju Island, Republic of Korea. Phylogenetic analysis of 16S rRNA gene sequences indicated that the two strains belonged to the genera Nocardioides and Hyunsoonleella , owing to their high similarities to Nocardioides jensenii DSM 29641 T (97.5 %) and Hyunsoonleella rubra FA042 T (96.3 %), respectively. These values are much lower than the gold standard for bacterial species (98.7 %). The average nucleotide identity values between strains MJB4 T , SJ7 T and the reference strains, Nocardioides jensenii DSM 29641 T , Nocardioides daejeonensis MJ31 T and Hyunsoonleella flava T58 T were 77.2, 75.9 and 75.4 %, respectively. Strains MJB4 T and SJ7 T and the type strains of the species involved in system incidence have average nucleotide identity and average amino acid threshold values of 60.1–82.6 % for the species boundary (95–96 %), which confirms that strains MJB4 T and SJ7 T represent two new species of genus Nocardioides and Hyunsoonleella , respectively. Based on phylogenetic and phenotypic data, strains MJB4 T and SJ7 T are considered to represent novel species of the genus Nocardioides and Hyunsoonleella , respectively, for which the names Nocardioides donggukensis sp. nov. (type strain MJB4 T =KACC 21724 T =NBRC 114402 T ) and Hyunsoonleella aquatilis sp. nov., (type strain SJ7 T =KACC 21715 T =NBRC 114486 T ) have been proposed.
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A yellow-coloured, Gram-negative, motile, strictly aerobic bacterial strain, designated strain DAC4T, was isolated from a soil sample collected at Ahnmok Beach (Busan, Republic of Korea). The cells of strain DAC4T were rod-shaped and the colonies that formed were round and convex. The results of phylogenetic analysis based on the 16S rRNA gene sequence of strain DAC4T revealed that the bacterium belongs to the genus Sphingomonas, family Sphingomonadaceae, and that it was most closely related to Sphingomonas jaspsi DSM 18422T (98.01 %), Sphingomonas rhizophila KACC 19189T (97.76 %), Sphingomonas mesophila KCTC 62179T(97.30 %), Sphingomonas sedimincola KCTC 12629T (97.16 %) and Sphingomonas oryziterrae KCTC 22476T (97.05 %). The major respiratory quinone was Q-10, and the major cellular fatty acids were summed feature 8 (C18 : 1ω7c) and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c). The whole genome DNA G+C content of strain DAC4T was 62.16 mol%. Phosphatidylethanolamine, diphosphatidylglycerol, sphingoglycolipids, phosphatidylglycerol, phosphatidylcholine, four undefined glycolipids and an undefined lipid were detected in strain DAC4T, and the strain had sym-homospermidine as a major polyamine. The in silico DNA-DNA hybridization and average nucleotide identity values between strain DAC4T and the closely related taxa S. jaspsi and S. mesophila were 75.5/23.5 % and 73.5 /18.5%, respectively. The fluorimetric DNA-DNA hybridization results showed that strain DAC4T and S. rhizophila, S. sediminicola and S. oryziterrae have 37.1, 35.2 and 32.2 % DNA similarity, respectively. Based on phylogenetic, phenotypic and chemotaxonomic distinctiveness, strain DAC4T (=KCTC 62107T=JCM 32377T) is classified as a novel species of the genus Sphingomonas, for which the name Sphingomonas edaphi sp. nov. is proposed.
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Spores of a thymine-requiring mutant of Bacillus subtilis 168 leucine(-), indole(-), thymine(-)) were uniformly labeled with (3)H-thymidine. These were seeded on thinlayer agar plates where they germinated into long-chained microcolonies. Autoradiograms were used to measure the distribution of labeled deoxyribonucleic acid in the chains of cells, which ranged in length from 2 to 32 cells. Four major grain clusters appeared in most chains. These clusters were homogeneous in size; their grain numbers were distributed symmetrically from 9 to 15 with an average of 12.0. When three or fewer major clusters appeared in short chains, some of them were composed of two subclusters. However, there were always four clusters per chain when these subclusters were counted as individuals. Groupings containing two to eight grains appeared, as well as the four major clusters in longer chains. These minor groups were fragments of the major clusters. In contrast to the symmetrical distribution of major clusters, fragmented clusters were distributed at random, indicating random fragmentation. The total number of major and minor clusters increased at a constant exponential rate when measured against total cell number per chain, i.e., number of generations. It was calculated from the rate that a detectable fragmentation, at least 16% of a conserved unit (defined as a single strand of the complete chromosome), occurred every 6.0 generations. These results led us to conclude that each B. subtilis spore contained four conserved units or two completed chromosomes. Segregation of the four units into progeny cells was almost random. The one notable exception was a conserved unit which frequently appeared in a terminal cell to which an empty spore coat was attached. The presence of two chromosomes in the spore is consistent with our proposed structure of the completed chromosome, in which two sister chromosomes are covalently linked at the initiation region. This double chromosome may be incorporated into the spore without further structural change.
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Excerpt INTRODUCTION Autoradiography of Escherichia coli, labeled with tritiated thymidine and lysed with duponol, has shown that the bacterial chromosome comprises a single piece of DNA which is probably duplicated at a single growing point (Cairns, 1963). Further, it seemed likely from the variety of structures seen that this DNA is in the form of a circle while it is being replicated, even though no intact replicating circles had, at that time, been found. There is no immediate prospect of proving that the bacterial chromosome is simply a continuous DNA double helix; the existence, for example, of protein linkers scattered along the chromosome (Freese, 1958) could be disproved only by a degree of purification of the intact chromosome that, at the moment, is technically impossible. So, rather than attempt any further purification, an effort was made to extract the chromosome as an intact but replicating circle by lysing labeled bacteria with...
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An unusual heavy form of DNA has been isolated from the spores of two wildtype strains of Bacillus subtilis, Marburg. This DNA is double-stranded and differs from the DNA isolated from exponentially growing cells by its higher buoyant density in caesium chloride equilibrium centrifugation and by its higher Tm. However, analysis of base composition has shown identical G + C content in both species of DNA. No abnormal bases or sugars other than deoxyribose have been found by the analytical methods used. No difference was found between the spore and vegetative DNA in either the alkaline dissociation curves or difference spectra. The heavy-spore DNA appears during sporulation shortly before the onset of thermal resistance in the bacterial population. Its specific content reaches a maximum 12 to 15 hours later. After germination, this DNA disappears during outgrowth sometime prior to the first cell division. The nature of the spore DNA and the cause of its physical-chemical changes are as yet unknown.
Article
A sample of triply marked pneumococcal DNA has been thermally denatured at pH 7 and ionic strengths of 0·1 M and 3 × 10−4M. The inactivation temperatures for the markers have been compared to the hyperchromicity midpoint and the marker densities reported by Rolfe & Ephrussi-Taylor (1961). We find that: (1) at low ionic strength, the transition breadth for inactivation is much greater than at high μ; (2) the inactivation midpoint temperatures at low μ are much greater than the ambient hyperchromicity Tm, while at high μ, this difference is small; (c) the differences between inactivation midpoints for different markers are increased at low μ. We suggest that the inactivation of each marker involves the denaturation of CG-rich critical regions. The critical regions for low ionic strength denaturation are shorter than the critical regions for high ionic strength denaturation. They are richer in GC content and more heterogeneous in base composition. We interpret the increased transition breadth at low ionic strength as evidence for a series of partially denatured states for each marker molecule.
Article
A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparations relatively free from protein and RNA. Alternative methods of cell disruption and DNA isolation have been described and compared. DNA capable of transforming homologous strains has been used to test various steps in the procedure and preparations have been obtained possessing high specific activities. Representative samples have been characterized for their thermal stability and sedimentation behaviour.
Article
The structure of renatured T4 DNA has been studied by CsCl density-gradient centrifugation. It has been found that the products of the reaction differ, depending on the method used for denaturation of the DNA. If denaturation is carried out without taking precautions to prevent chain degradation, for example, by heat, the DNA formed by renaturation shows approximately 70% recovery of the native structure as judged by its density. With long times of annealing, the DNA can recover the native density. This behavior is also observed with bacterial DNA samples. On the other hand, if precautions arc taken to prevent chain degradation during denaturation, two products appear as a result of renaturation. One of them is undistinguishable from native T4 DNA, whereas the second one consists of highly aggregated DNA which shows only a partial recovery of the native structure. With long times of annealing, this second species recovers the native density but retains its highly aggregated nature. At higher ionic-strengths, renaturation follows a different pattern and a single product is formed. The relevance of all these observations to the kinetic anomalies reported in the previous communication is discussed.
Article
The previously discovered linear relation between the base composition of DNA, expressed in terms of percentage of guanine plus cytosine bases, and the denaturation temperature, Tm, has been further investigated. By means of measurements on 41 samples of known base composition the previously observed relation has been confirmed. It can be summarized thus : for a solvent containing 0·2 M-Na+, Tm = 69·3 + 0·41 (G-C) where Tm is in degrees Centigrade and G-C refers to the mole percentage of guanine plus cytosine. The deviations of experimental points from this relation are no more than that expected from the uncertainties of base analysis and the variations of a half degree in the reproducibility of determining the Tm. Consequently it appears that the measurement of the Tm is a satisfactory means of determining base composition in DNA. The Tm values are most simply measured by following the absorbance at 260 mμ as a function of temperature of the DNA solution and noting the midpoint of the hyperchromic rise. Only 10 to 50 μg of DNA are required.A number of other DNA samples of unknown base composition have been examined in this manner and their base compositions recorded.
Article
It is suggested that the replication of the bacterial genome during the division cycle of Escherichia coli growing with doubling times between approximately 20 and 60 minutes can be described by two constants: C, the time for a replication point to traverse the genome, and D, the time between the end of a round of replication and cell division. Direct measurements of C, D and C + D agree with the model. The predictions of the model are consistent with autoradiographic and biochemical data, and with the finding of “gaps” in DNA synthesis during slow growth and multiple replication forks during rapid growth. Measurements of the DNA content of cells with predicted chromosome configurations led to an estimate of the molecular weight of the bacterial genome (non-replicating) of 2.6 × 109 daltons, in good agreement with other, independent measurements.
Article
The conditions that produce the optimal re-formation of the native DNA conformation from denatured DNA have been examined. The restoration of the native conformation, called thermal renaturation, has been found to depend markedly on the source of the DNA; mammalian DNA coming from cells with very large DNA content renatures only slightly, bacterial DNA with greatly reduced DNA content per cell undergoes extensive renaturation, and the very smallest bacteria together with bacteriophage, having the lowest DNA contents, show nearly complete renaturation. With a given DNA, the optimal renaturation was found to occur at about 25° below the denaturation temperature, Tm. The extent of renaturation was optimal above 0·4 M-Na+ and increased with molecular weight. The identity of the renatured DNA and the native material can be shown in two ways: the similarity of the absorbance-temperature curves and the similarity of the rate of thermal inactivation of biological markers at temperatures somewhat above Tm. This reproduceability of the helix-coil transition and the course of thermal inactivation demonstrates that the same secondary structure has re-formed and that non-specific hydrogen bonding is not involved.
Article
The DNA content of the Bacillus subtilis spore has been redetermined and found to agree with the previous estimate by Fitz-James & Young (1959). This sets an upper limit of 3·0 × 109 daltons (1700 μ of the B form) as the amount of DNA in the B. subtilis genome. By autoradiography continuous chromosomal lengths of 800 to 900 μ are shown to occur in vegetative cells. From recent estimates of the size of the genome, these results suggest that it exists as a single chromosome, two of which are present in the spore. However, considering the fragility of the bacterial chromosome, the present results do not rule out the possibility that the B. subtilis genome exists as a much longer chromosomal structure, only one of which is present in the spore.
Article
A new method is proposed to measure relatedness amongst bacteria, based on renaturation rate determinations of DNA types and their mixture. The equations, relating the degree of binding with renaturation rates, are calculated for several cases. The optimal conditions for measurements are given. The method was checked in several ways. The advantages are summarized.
Article
Reciprocal hybridizations were carried out between DNA fromPseudomonas fluorescens, P. putida and a xanthomonad,P. campestris var.pelargonii. Furthermore, DNA-fragments from each organism, preselected by hybridization with each of the other two strains, were again hybridized with all three DNA-types. The molecular weight of the chromosomal DNA from the three organisms is about equal, having a value of 2.4·0.4×109 daltons/nucleoid or 3.9 × 106 nucleotide pairs/nucleoid. About half of the DNA from each organism has a similar, but not identical, nucleotide sequence. Theputida- andfluorescens-DNA share an additional 33°. homology. From the present and previous experiments it can be hypothesized that all xanthomonads share an additional stretch of homologous DNA, amounting to some 25–40% of the bacterial chromosome. The remaining DNA in each organism (about 15%) is probably responsible for strain individuality (varieties, races and strains within the same genospecies). The results suggest that the three organisms are derived from a common pool of ancestors; that the xanthomonads diverged first or fastest and that the split betweenP. putida andP. fluorescens is a more recent event. The mean molar (guanine + cytosine) content of the common part is identical to that of the total parent DNA. The average value for the three organisms is 62·2% (G+C). The direction of the evolutionary drift of the (G+C) content between these closely related organisms is not detectable.
Article
The rate of renaturation of fully denatured DNA is kinetically a second-order reaction. The reaction rate increases as the temperature decreases below Tm†, reaching a broad flat maximum from 15 to 30 °C below Tm and then decreases with a further decrease in temperature. Let N be the complexity of the DNA or the number of base-pairs in non-repeating sequences per virus or cell for the given DNA, and L the average number of nucleotides per single strand of the denatured DNA preparation. Then, the second-order renaturation rate constants for all DNA's are given approximately by . mole−1 sec−1 at (Tm − 25) °C and at [Na+] = 1.0 mole 1.−1 in aqueous solution. The reaction rate increases slightly with the GC content of the DNA. The reaction rate at the temperature maximum (Tm − 25) °C is inversely proportional to solvent viscosity, when the viscosity is changed by the addition of components which either have a small (sucrose, glycerol, ethylene glycol) or a large (NaClO4) effect on Tm. It is proposed that the mechanism of the reaction involves the joining of short, homologous sites on the two strands followed by a fast, reversible zippering reaction with forward rate constant kt. A computer analysis for this model explains the temperature and the GC dependence. To explain the viscosity dependence it is proposed that kf is inversely proportional to viscosity; that is, the zippering reaction is hydrodynamically limited. Any simple theory predicts ; the observed L0.5 length dependence is attributed to an excluded volume or steric hindrance effect, that is, to restricted interpenetration of the two complementary denatured DNA coils.
Article
A mathematical method is given for calculating the upper limit of possible DNA homology between any set of two bacteria; it depends on three parameters: the mean nucleotide composition, the molecular weight and the compositional nucleotide distribution of chromosomal DNA. The theoretical predictions are checked and confirmed by comparison with available experimental DNA hybridization data. The standard deviation of the compositional nucleotide distribution (expressed as molar per cent guanine + cytosine) of chromosomal DNA, was calculated for each of some 2500 different bacteria, belonging to 36 genera. The values range from 3 to about 5.5% GC, using an improved correction factor. When the mean nucleotide compositions of bacteria differ by at least 18 to 30% GC, there are almost no common nucleotide sequences and the organisms are evolutionarily far removed from each other and taxonomically unrelated.
Article
The kinetics of renaturation of heat- or formamide-denatured DNA have been studied by following the change of optical density at a constant temperature. Solvents of different ionic strength and various DNA samples have been used. At the lower ionic strengths studied, the reaction follows second-order kinetics, substantiating the hypothesis that strands of native DNA separate upon denaturation and recombine during renaturation. As the ionic strength is increased at a constant temperature, the reaction deviates from simple second-order behavior. This appears to be the result of the inhibition to rewinding caused by short helical segments in the denatured DNA which are more stable at the higher ionic strenth.
Article
Cell mass, the average number of nuclei/cell and the content of RNA and DNA were studied in Salmonella typhimurium during balanced (steady state) growth in different media. These quantities could be described as exponential functions of the growth rates afforded by the various media at a given temperature. The size and chemical composition characteristic of a given medium were not influenced by the temperature of cultivation. Thus, under conditions of balanced growth, this organism exists in one of a large number of possible stable physiological states. The variations in mass/cell are due to changes in the number of nuclei/cell as well as in mass/nucleus. An increase in the number of ribonucleoprotein particles at higher growth rates could, it appears, largely account for the increase in mass/nucleus. Calculations indicate that the rate of protein synthesis per unit RNA is nearly the same at all growth rates.
Article
The ultraviolet absorption spectra of a number of DNA's of natural origin, and varying in base composition between 73 and 37 mole per cent (guanine plus cytosine) have been obtained in a methanolic medium of low ionic strength both at room temperature and at a temperature 10°C above the midpoint for the helix → coil transition. In addition, the spectra of the stoicheiometric mixtures of deoxynucleotides corresponding to the identical base composition have been computed from published data. Similar sets of spectra have also been obtained for the synthetic DNA's, dAT and dG : dC. From these spectra we have calculated two sets of difference spectra (coils-helix) and (nucleotides-helix), as well as the corresponding wavelength-dependent hypochromicities as a function of base composition. The results have been interpreted in terms of a linear combination of difference spectra for isolated “average” A-T and G-C pairs in the helix, which differ from those of the corresponding synthetic, helical polydeoxyribonucleotides.
Article
The nuclear DNA complement of Hemophilus influenzae has been determined to be 700 to 800 × 106 daltons. An isolation procedure has been developed which gives high yields of DNA with a maximum molecular weight of 400×106, or one-half of the nuclear DNA complement. Linkage of previously unlinked markers has been obtained in transformation assays.The isolation procedure has been extended to Escherichia coli and Serratia marcescens.
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