Brief Report, 2002_A Fluorimetric Method for the Estimation of G+C Mol% Content in Microorganisms by Thermal Denaturation Temperature

8/3/2019 Brief Report, 2002_A Fluorimetric Method for the Estimation of G+C Mol% Content in Microorganisms by Thermal D

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Environmental Microbiology (2002) 4(11), 770 773

2002 Blackwell Science Ltd

Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Blackwell Science, 20024771774Short communicationFluorimetric determination of %GCJ.M. Gonzalez and C.Saiz-Jimenez

Received 1 August, 2002; accepted 10 September, 2002. *For corre-spondence. E-mail [email protected]; Tel. (+34) 954624 711;Fax (+34) 954624 002.

Brief report

A fluorimetric method for the estimation of

G+C mol% content in microorganisms by thermaldenaturation temperature

J. M. Gonzalez* and C. Saiz-Jimenez

Instituto de Recursos Naturales y Agrobiologia, CSIC,

Apartado 1052, 41080 Sevilla, Spain.

Summary

G+C mol% content in microorganisms is one of

the recommended characteristics for the standarddescription of bacterial species. In this study we

present a novel fluorimetric method to estimate

the G+C mol% content in microorganisms. Double-

stranded DNA was specifically stained with SYBR

Green I, and its thermal denaturalization was followed

by measuring a decrease in fluorescence using a

real-time PCR thermocycler. Unlike most previous

determinations of G+C mol%, in this study only DNA

from microorganisms with an available completely

sequenced genome were used to prepare the calibra-

tion curves. Calibration curves showed a linear rela-

tionship between G+C mol% content and meltingtemperature and they were performed both in the

absence and presence of 30% formamide. This pro-

tocol proves to be a rapid and inexpensive method to

estimate DNA base ratios of novel microorganisms.

Introduction

The information contained in the DNA is encoded by four

nitrogenated bases A, C, G and T (adenine, cytosine,

guanine and thymine respectively). The proportion of

G+C, or DNA base ratio (moles per cent of G+C), is

considered part of the standard description of bacterial

taxa (Vandamme et al., 1996; Rosell-Mora and Amann,

2001). Mol% G+C content varies between 24 and 76% in

the bacterial world (Torsvik et al., 1995; Vandamme et al.,

1996).

Several techniques have been used for assessing the

fraction of G+C in the genome of microorganisms. A direct

method based on the sequencing of the whole genome

from the query microbial species is a straightforward pro-

cedure; however, this would represent a long and unfea-

sible project only for the purpose of obtaining the G+C

fraction in the DNA of a microbial species. The two most

common approaches are either by high-performance liq-

uid chromatography (HPLC) or by thermal denaturaliza-tion techniques. High-performance liquid chromatography

techniques are usually considered accurate but require

the set up of a HPLC system specifically for this purpose

which is expensive and only worthwhile if these determi-

nations are performed with high frequency (Tamaoka and

Komagawa, 1984). Measurements of absorbance during

the thermal denaturalization of DNA have been used

as a rapid alternative to estimate the content of G+C

in genomic DNA from microorganisms. This strategy

requires the availability of a spectrophotometer with a

thermal controller. Melting temperatures of DNA mole-

cules and their percentages of G+C follow a linearrelationship (Marmur and Doty, 1962; De Ley et al.,

1970). Fluorimetric determinations of DNA denaturaliza-

tion should translate in a sensitive and simple method

for assessing G+C mol% content in microorganisms.

Because of the widespread use of quantitative poly-

merase chain reaction (PCR) techniques, real-time PCR

thermocyclers are becoming common in most laborato-

ries. Herein, we propose an easy, rapid, high-throughput,

fluorimetric technique to estimate percentage G+C con-

tent in DNA samples. The method uses a fluorescent,

double-strand specific dye and the melting temperature

software and hardware capabilities of modern quanti-

tative, real-time PCR thermocyclers. We obtained

calibration relationships between GC mol% and melt-

ing temperature using microorganisms with completely

sequenced genomes.

Results and discussion

Most modern real-time PCR thermocyclers allow to per-

form melting curve experiments. We used an iQ iCycler


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Fluorimetric determination of %GC 771

2002 Blackwell Science Ltd, Environmental Microbiology, 4, 770773

real-time thermocycler (Bio-Rad, Hercules, CA) to obtain

melting curves of genomic DNA from a number of micro-

organisms and calculated their melting temperatures.

Eleven prokaryotic strains with completely sequenced

genomes were used in this study (Table 1). The G+C

mol% of these strains ranged from 30.9% to 66.6%. Melt-

ing temperatures were calculated in the presence and

absence of 30% formamide. In the absence of formamide,

melting temperatures for the microbial species used in this

study ranged between 70C and 90C (Table 1). In the

presence of 30% formamide the range of melting temper-

atures for the microorganisms used in this study was

approximately between 50C and 70C (Table 1).

SYBR Green I (Molecular Probes, Eugene, Oregon) is

a fluorescent dye showing high fluorescence when it binds

to double-stranded DNA. Thus, using this dye, the double-

stranded DNA molecules can be exclusively quantified

during the denaturalization experiments. SYBR Green I

shows maximum fluorescence at excitation and emission

wavelengths of 497 and 520 nm respectively. These

peaks are coincident with the FAM filter set availablein the standard configuration of any real-time PCR

thermocycler.

The G+C mol% from these microorganisms showed a

positive relationship with the melting temperature (Tm) of

their total genomic DNAs (Fig. 1). In the absence of for-

mamide, the obtained regression line (n= 11, r2= 0.99,

P


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772 J. M. Gonzalez and C. Saiz-Jimenez


between G+C mol% and melting temperature is an impor-

tant factor for accurate G+C mol% results from thermal

denaturation curves. The use of HPLC techniques for

estimates of the DNA base ratio (Tamaoka and

Komagawa, 1984; Meshbah et al., 1989) provide accurate

results although the method involve the use of a dedicated

HPLC set up not available in non-specialized laboratories.

A relationship between G+C mol% content and meltingtemperature is useful for estimating percentage GC con-

tent of novel microorganisms during their taxonomic clas-

sification and is universal for the Prokaryotes (De Ley

et al., 1970). A previous G+C mol% versus melting tem-

perature relationship gave a regression coefficient of 2.44

(De Ley et al., 1970). This slope is significantly higher than

the one resulting from our study and these differences

might be a consequence of the percentage GC values

estimated for the species used in the calibration (see

above paragraph) and the methods employed to obtain

the melting curves; former studies measured absorbance

using automatic recording thermal spectrophotometers

(De Ley et al., 1970) whereas we have measured fluores-

cence emitted by a double-stranded DNA-specific dye on

a real-time PCR thermocycler. A previous percentage GC

versus melting temperature calibration curve (De Ley

et al., 1970) would imply the possibility of melting curves

reaching temperatures over the boiling point of water for

microorganisms with high GC content (>70%). In this

study, we have showed that the G+C mol% estimates for

these microorganisms with extremely high percentage GC

can also be performed in the presence of 30% formamide.

The presence of 30% formamide decreased the melt-

ing temperature significantly (by about 18C; Fig. 1)

(Vandamme et al., 1996). Thus, %G+C estimates from

thermal denaturation curves may be performed for any

Prokaryote.

Prokaryotic taxonomists agree that a reliable classifica-

tion of a microorganism can only be achieved through itsstudy by a number of different techniques in what is gen-

erally known as the polyphasic approach (Vandamme

et al., 1996). This implies that both the genomic informa-

tion and the phenotype of a microorganism should be

investigated. The DNA base ratio was the first genomic

technology applied to prokaryote taxonomy (Lee et al.,

1956) and has proved to be a useful and routine approach

of distinguishing between species with a similar pheno-

type (Goodfellow and ODonnell, 1993). Empirically, it has

been shown that organisms that differ by more than

10 mol% do not belong to the same genus and that spe-

cies are within a range of 5 mol% (Wayne et al., 1987).

Well-defined species usually narrow this range to 3%

(Stackebrandt and Liesack, 1993; Vandamme et al.,

1996). Species comparisons using the DNA base ratio

can only be used to discriminate between different strains,

as similar DNA base ratios do not necessarily imply phy-

logenetic similarity (Rosell-Mora and Amann, 2001).

Today, the DNA base ratio, or mol% G+C content, is one

of the recommended characteristics for the standard

description of prokaryotic species. This study proposes an

Fig. 1. A. Calibration curve of G+C mol% versus melting temperature (Tm) for a number of microorganisms with their genomic sequence available.B. A calibration curve of the same strains in the presence of formamide (30%) is also shown.


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Fluorimetric determination of %GC 773


easy, rapid, and inexpensive method for estimating the

G+C mol% of microorganisms, which could be performed

by any researcher interested in the classification of novel

microorganisms.

Acknowledgements

The authors thank Drs Luz Candenas de Lujan and FranciscoPinto for the use of the iQ iCycler and their valuable

assistance and comments. We are greatly appreciated to

the CECT (Spanish Culture Collection) for providing with

the bacterial species needed for the calibration curves. The

authors acknowledge funding from the European projects

COALITION (EVK4-CT-199920001) and CATS (EVK4-

CT-200000028). J.M.G. thanks funding from the Spanish

Ministry of Science and Technology, Ramn y Cajal

programme.

References

Charbonnier, F.P., and Forterre (1989) Purification of plas-

mids from thermophilic and hyperthermophilic Archaea. InArchaea, a Laboratory Manual Thermophiles Robb, F.T.,

and Place, A.R., (eds), Cold Spring Harbor, New York: Cold

Spring Harbor Laboratory Press, pp. 8790.

De Ley, J., Cattoir, H., and Reynaerts, A. (1970) The quan-

titative measurement of DNA hybridization from renatur-

ation rates. Eur J Biochem12: 133142.

Fiala, G., and Stetter, K.O. (1986) Pyrococcus furiosus sp.

nov. represents a novel genus of marine heterotrophic

archaebacteria growing optimally at 100C. Arch Microbiol

145: 5661.

Gonzlez, J.M., Masuchi, Y., Robb, F.T., Ammerman, J.W.,

Maeder, D.L., Yanagibayashi, M., et al. (1998) Pyrococcus

horikoshiisp. nov., a hyperthermophilic archaeon isolated

from a hydrothermal vent at the Okinawa Trough. Extremo-philes2: 123130.

Goodfellow, M., and ODonnell (1993) Roots of bacterial

systematics. In Handbook of New Bacterial Systematics

Goodfellow, M., and ODonnell, A.G., (eds), London:

Academic Press, pp. 354.

Lee, K.Y., Wahl, R., and Barbu, E. (1956) Contenu en bases

purique et pyrimidiques des acides deoxyribonucleiques

des bacteries. Ann Inst Pasteur91: 212224.

Marmur, J. (1961) A procedure for the isolation of deoxyribo-

nucleic acid from microorganisms. J Mol Biol3: 208218.

Marmur, J., and Doty, P. (1962) Determination of the base

composition of deoxyribonucleic acid from its thermal dena-

turation temperature. J Mol Biol5: 109118.

Meshbah, M., Premachandran, U., and Whitmann, W. (1989)

Precise measurement of the G+C content of deoxyribonu-

cleic acid by high performance liquid chromatography. Int

J Syst Bacteriol39: 159167.

Rosell-Mora, R., and Amann, R. (2001) The species concept

for prokaryotes. FEMS Microbiol Rev25: 3667.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual, 2nd edn. Cold Spring

Harbor, New York: Cold Spring Harbor Laboratory.

Sokal, R.R., and Rohlf, F.J. (1981) Biometry, 2nd edn. New

York: W.H. Freeman.

Stackebrandt, E., and Liesack (1993) Nucleic acids and

classification In Handbook of New Bacterial Systematics.

M. Goodfellow and A.G.ODonnell, (eds). Academic Press,London, pp. 151194.

Tamaoka, J., and Komagawa, K. (1984) Determination of

DNA base composition by reversed-phase high-

performance liquid chromatography. FEMS Microbiol Lett

25: 125128.

Torsvik, V.F.L., Daae and Goksyr (1995) Extraction, purifica-

tion, and analysis of DNA from soil bacteria. In Nucleic

Acids in the Environment: Methods and Applications.

Trevors, J.T., and van Elsas, J.D., (eds). New York: Springer

Verlag, pp. 2948.

Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, K.,

and Swings, J. (1996) Polyphasic taxonomy, a consensus

approach to bacterial systematics. Microbiol Rev60: 407

438.Wayne, L.G. et al. (1987) Report of the ad hoc committee on

reconciliation of approaches to bacterial systematics. Int J

Syst Bacteriol37: 463464.


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Brief Report, 2002_A Fluorimetric Method for the Estimation of G+C Mol% Content in Microorganisms by Thermal Denaturation Temperature