A rapid chemical procedure for isolation and purification of chromosomal DNA from gram-negative bacilli

doi:10.1016/0003-2697(87)90005-4

Analytical Biochemistry

Volume 162, Issue 1, April 1987, Pages 18-23

https://doi.org/10.1016/0003-2697(87)90005-4 Get rights and content

Abstract

A rapid and simple method for preparing chromosomal DNA from gram-negative bacilli is presented. It is based on the alkaline (NaOH 0.03 m) lysis of cell walls. The resulting emulsion is purified by proteinase K (0.625 mg/g of wet wt), SDS, and the deproteinizing agent (chloroform isoamyl alcohol). The purity, molecular nature, and yield of DNA obtained by the present method are compared with those of DNA extracted by Marmur's procedure and a Marmur's modified procedure. We have developed and standardized this original method to isolate double-stranded DNA, free of proteins and RNA contamination and with a significantly higher yield of DNA than the two other methods. This procedure is particularly useful for strains with low growth and can be applied in every field concerned with DNA analysis.

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Citation Excerpt :
Bacterial DNA was extracted from all the isolates after overnight growth in LB Broth at 30 °C. DNA from Gram positive bacteria was extracted using the InstaGene Matrix (Bio-Rad, Milan, Italy) according to the manufacturer’s instructions, while from Gram negative bacteria DNA it was extracted by alkaline lysis as previously described by Beji et al. (1987). In order to reduce the number of isolates to be identified by partial 16S rRNA gene sequencing, the extracted DNA was subjected to Randomly Amplified Polymorphic DNA–Polymerase Chain Reaction (RAPD–PCR) for biotype screening among the isolates.
The aim of this study was to study the psychrotrophic microbiota developing during milk creaming of Grana Trentino cheese-making. 138 isolates from raw whole milk, cream and skim milk samples were screened by Randomly amplified polymorphic DNA PCR biotyping and representative strains of each biotype were characterised by partial 16S rRNA gene sequencing and enzymatic activity. Pseudomonadaceae were commonly isolated in cream samples while Streptococcaceae and Enterobacteriaceae in milk samples. Moraxellaceae and Flavobacteriaceae were found in both cream and milk samples.
More than 80% of psychrotrophic isolates could grow at 37 °C. All Flavobacteriaceae and half of Pseudomonadaceae biotypes displayed proteolytic activity on milk agar even at low temperatures such as 10 °C. All Streptococcaceae and some of Enterobacteriaceae displayed acidifying activity and almost all Acinetobacter spp. (Moraxellaceae) displayed lipolytic activity towards tributyrin.
Even if psychrotrophic bacteria is not the dominant microbial group in raw milk, their total number increases during creaming and becomes one of the most present group together with Lactic Acid Bacteria. Their enzymatic activities may be key players in determining milk quality for cheese making.
Pseudomonas brenneri sp. nov., a new species isolated from natural mineral waters
2001, Research in Microbiology
The vernacular name ‘fluorescent Pseudomonas group 97-391’ was coined for a group of 11 strains isolated from two French natural mineral waters. All these strains were Gram-negative, rod-shaped and motile by means of a single polar flagellum. They produced fluorescent pigment (pyoverdin) on King B medium, catalase and cytochrome oxidase. They were not able to accumulate poly-β-hydroxybutyrate. They were capable of respiratory but not fermentative metabolism. DNA–DNA hybridization results and DNA base composition analysis revealed that strains of the ‘fluorescent Pseudomonas group 97-391’ were members of a new species, for which the name Pseudomonas brenneri sp. nov. (type strain CIP 106646^T) is proposed. The levels of DNA–DNA relatedness within this group ranged from 70 to 100% with ΔT_m below 1°C. The G+C content of the DNA of the type strain was 58 mol%. DNA relatedness with 72 strains representing well-known or partially characterized species of the genus Pseudomonas (sensu stricto) was below 48%. The complete 16S rRNA sequence of the type strain CIP 106646^T was determined and compared with those of the type strains of Pseudomonas species. Finally, a phylogenetic tree was inferred from sequence analysis and demonstrated that the new species fell into the ‘Pseudomonas fluorescens intrageneric cluster’. The clinical significance of P. brenneri is unknown.
Taxonomic study of bacteria isolated from Lebanese spring waters: Proposal for Pseudomonas cedrella sp. nov. and P. orientalis sp. nov.
1999, Research in Microbiology
Deoxyribonucleic acid relatedness studies (S1 nuclease method) have shown that 15 strains isolated from three Lebanese spring waters, belonging to the genus Pseudomonas, formed two homogeneous DNA groups, with a within-group DNA relatedness ranging from 70 to 100%. These groups are referred to as Pseudomonas cedrella sp. nov. and Pseudomonas orientalis sp.nov. These strains were previously grouped on the basis of a numerical analysis in phenons Ve, Vd, Vg, and VI. DNA relatedness with 65 strains representing 24 species of the genus Pseudomonas sensu stricto was below 50%. The highest DNA binding value (50%) was found with P. marginalis species. A comparison of the complete 16S rRNA gene sequences of the strains representing the two new deoxyribonucleic acid hybridization groups, i.e., strains CFML 96–198^T and CFML 96–170^T, and the sequence of other strains of the genus Pseudomonas revealed that these strains (CFML 96–198^T and CFML 96–170^T) fell within the'Pseudomonas fluorescens intrageneric cluster'. The G+C contents of the DNA of P. cedrella CIP 105541^T and P. orientalis CIP 105540^T were 59 and 60 mol%, respectively. The two species can be differentiated from each other by the fact that P. cedrella strains hydrolyze erythritol and D-lyxose. P. cedrella grouped together a total of nine strains from phenotypic groups Ve, Vg, and VI. P. orientalis grouped together six strains from both phenotypic groups Vd and Ve.
Étude taxinomique des souches bactériennes isolées d'eaux de source libanaises : proposition de Pseudomonas cedrella sp. nov et P. orientalis sp. nov. L'hybridation ADN-ADN (méthode à la nucléase S1) a montré que 15 souches, isolées de trois eaux de sources libanaises, appartenant au genre Pseudomonas, forment deux groupes génomiques avec un taux d'hybridation au sein du groupe allant de 70 à 100 %. Ces deux groupes sont nommés P. cedrella sp. nov. et P. orientalis sp. nov. Ces souches étaient antérieurement groupées sur la base d'une analyse numérique dans les phénons Ve, Vd, Vg et VI. Le taux d'hybridation ADN-ADN avec 65 souches représentant 24 espèces du genre Pseudomonas sensu stricto est inférieur à 50 %. La valeur la plus élévée est notée avec l'espèce P. marginalis. La comparaison des séquences de l'ARNr 16S des souches représentant les deux groupes génomiques (CFML 96–198^T et CFML 96–170^T) avec les autres séquences des espèces du genre Pseudomonas montre que ces deux souches se positionnent dans le groupe intragénique de « P. fluorescens ». Le GC% de P. cedrella CIP 105541^T et de P. orientalis CIP 105540^T est de 59 et 60 mol% respectivement. Les deux espèces peuvent être différenciées entre elles puisque P. cedrella hydrolyse l'érythritol et le D-lyxose. P. cedrella regroupe neuf souches provenant de trois groupes phénotypiques, Ve, Vg, et VI. P. orientalis regroupe six souches de deux groupes phénotypiques, Vd et Ve.
Taxonomic study of bacteria isolated from natural mineral waters: Proposal of Pseudomonas jessenii sp. nov. and Pseudomonas mandelii sp. nov.
1999, Systematic and Applied Microbiology
The taxonomic position of 23 strains isolated from mineral waters and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster IX, subclusters XIIIa and XIIIc of Verhille, S., Elomari, M., Coroler, L., Izard, D., Leclerc, H. (Syst. Appl. Microbiol. 20, 137–149, 1997) has been genotypically further studied in the present work. On the basis of hybridization results, these strains were gathered into two new genomic groups for which we propose the names of Pseudomonas jessenii sp. nov. (Type strain CIP 105274^T) and Pseudomonas mandelii sp. nov. (Type strain CIP 105273^T). Deoxyribonucleic acid relatedness levels showed homologies ranging from 78 to 100% for Pseudomonas jessenii and from 77 to 100% for Pseudomonas mandelii. Furthermore, hybridization rates with 66 representative well characterized species or only partially characterized species of the genus Pseudomonas were below 53%, with ΔT_m values of 7 °C and more. The mol% G+C content ranged from 57 to 58. The two new species presented basic morphological characteristics common to all pseudomonads. Various phenotypic features, such as denitrification, growth at 4 °C or 41 °C, trigonel-line assimilation, α-L-glutamyl-L-histidine arylamidase activity, growth on benzoate and meso-tartrate were found to differentiate Pseudomonas jessenii from Pseudomonas mandelii and from other Pseudomonas species. Pseudomonas jessenii encompassed a total of 9 strains from both phenotypic groups IX and XIIIa. Pseudomonas mandelii clustered a total of 13 strains from both phenotypic groups IX and XIIIc. Their clinical significance is unknown. The 16S rDNA of each type strain was sequenced and compared with the known sequences of the representative strains of the genus Pseudomonas. A phylogenetic tree was constructed to determine the intrageneric relationships within the genus Pseudomonas.
A study of the fate of the autochtonous bacterial flora of still mineral waters by analysis of restriction fragment length polymorphism of genes coding for rRNA
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From five French springs producing “still” waters, 36 water samples each were aseptically taken at three stages: at the source, just before bottling and upon bottling. Specimens were examined immediately after drawing and periodically during storage at ambient temperature for one year. Monitoring included (i) microscopic examination, combining acridine orange epifluorescence cell count with an INT stain; (ii) enumeration of colony forming units on R₂A agar, incubated at 22 °C for 2 weeks. Colonies obtained on this medium were examined by a Gram stain, and for mobility, mode of attack on glucose, oxidase reaction, pigment production, in an API 20NE gallery, and restriction fragment length polymorphism profiles of RNA regions (“ribotyping”).
In the pristine samples AODC counts varied between 10³ and 10⁴ ml^-1, with INT+ cells varying from 10¹ to 10² ml^-1, and colony counts from 1 to 10 ml^-1. Upon storage for one week AODC counts reached the order of 10⁵ ml^-1, INT+ cells < 10⁴ ml^-1 and cfu's 10³-10⁴. These levels did not appreciably change upon further storage, except for INT+ counts which fluctuated somewhat, but never exceeded cfu numbers. Amongst 890 isolates from R₂A-agar, originating from all specimens, only 18% could be identified as: Ps. fluorescens > Brevundimonas vesicularis > Sphingomonas paucimobilis > Comamonas testosteroni > C. acidovorans. From an occasional spring other non-fermentative Gram negative rods were isolated sporadically.
The 890 isolates provided 378 different ribopatterns. Dependent on the spring, 44–114 different patterns were obtained per source. Dice coefficients of clustering varied from 20 to 95% similarity. Relative abundance of ribotypes in the different springs varied, some clusters being more specific for a given spring than others. Per source the incidence of repeatedly isolated types varied from 55–74%. Similarity of ribotypes recovered from the same source before and after bottling averaged 70%. These results suggest that a high percentage of closely related, if not indistinguishable, ribotypes of non-fermentative Gram negative rod-shaped bacteria occurring in pristine minerale waters is preserved throughout bottling and storage.
Pseudomonas rhodesiae sp. nov., a new species isolated from natural mineral waters
1996, Systematic and Applied Microbiology
Deoxyribonucleic acid relatedness studies (S1 nuclease method) showed that 7 strains of the cluster IIa previously described by Elomari et al. (J. Appl. Bacteriol 78, 71-81, (1995)) formed a homogenous deoxyribonucleic acid hybridization group. This group of strains forms a new species: Pseudomonas rhodesiae sp. nov. A total of 67 strains representing known or partially characterized sprecies of the genus Pseudomonas had 7 to 48 % DNA hybridization with Pseudomonas rhodesiae. This new species composed of strains isolated from natural mineral waters, were Gram negative, rod shaped, and motile by means of a single polar flagellum. They were not able to accumulate poly-γ-hydroxybutyrate, were capable of respiratory but not fermentative metabolism. They grew at 4°C but not a 41°C, produced fluorescent pigment, catalase, cytochrome oxidase and lecithinase, and possessed arginine dihydrolase system. They did not hydrolyse gelatin and starch, and were able to use glucose, trehalose, 2-ketogluconate, inositol, L-valine and γ-alanine. They possessed L-pyrrolidone arylamidase, L-histidyl-L-serine arylamidase but did not have osidase, esterase = C₁₂, esterase = C₁₄, esterase = C₁₆, and glycyl-L-tryptophane arylamidase. The average guanine + cytosine (G + C) content of the deoxyribonucleic acid was 59 ± 1 mol%. The type strain of P. rhodesiae has been deposited as CIP 104664¹. The clinical significance of Pseudomonas rhodesiae is unknown.

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A rapid chemical procedure for isolation and purification of chromosomal DNA from gram-negative bacilli

Abstract

J. Biol. Chem

J. Biol. Chem

Gene

Biophys. J

Annu. Rev. Microbiol

Amer. Soc. Microbiol

J. Mol. Biol

Microbios

J. Biochem