pPHL7 introduced into manmade closed aquatic microcosms

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SCIENCE

DIRECT-

doi: 10.1016/SO273-1177(03)00118-2

SURVIVALAND ALTERATION OF THE PLASMID-CONTAINING MICROORGANISM ESCHERICHIA COLI Z905/pPHL7 INTRODUCED INTO MANMADE CLOSED AQUATIC MICROCOSMS A.N. Boyandin, T.I. Lobova, L.Yu. Popova, N.S. Pechurkin Institute of Biophysics, Russian Academy of Sciences, Siberian Branch. Akademgorodok, Krasnoyarsk, 660036, Russia

ABSTRACT

It has been demonstrated that the transgenic microorganism Escherichia coli Z9OYpPHL7 (ApZux’) can exist for a long time at an elevated concentration of mineral salts. The microorganism was introduced into microcosms with sterile brackish water (salinity variable from 21 to 22 g I-‘) taken from Lake Shira (Khakasia, Russia). The surviv,l of the microorganism was estimated both by measuring the growth of the colonies on solid nutrient media and by the bioluminescence exhibited by the transgenic strain in samples from the microcosms and in the enrichment culture with the added selective factor - ampicillin (50 pg/ml). In the enrichment culture, the bioluminescent signal was registered through the 16Oday experiment. It has been shown that in the closed microcosms with brackish water the E. coli strain becomes heterogeneous in its ampicillin resistance. The populations of the transgenic strain were mainly represented by isolates able to persist in the medium containing 50 @ml, but there were also the cells (about 10%) with the threshold of ampicillin resistance not more than 0.05 @ml. Thus, it was shown that in the microcosms with brackish water and in the absence of the selective factor the transgenic strain survives and retair. the recombinant plasmid. 0 2003 COSPAR.Published by Elsevier Science Ltd. All rights reserved.

INTRODUCTION

Since genetically modified microorganisms (GMMOs) are used in biotechnology, it is necessary to conduct investigations to determine the fate of these organisms under various environmental conditions. It is essential to study the survivability of these organisms in ecosystems of various types and potential migration of recombinant structures among natural microbial populations. It is also important to conduct thorough monitoring of both the GMMO cells and the cloned genetic material. Besides the classical methods of monitoring, based on direct isolation of microorganisms from samples of natural materials, and molecular methods, these investigations can employ genes of bioluminescent systems, cloned in transgenic strains. Lux-genes can be used to study such parameters as the persistence of the transformed genome, its regulation, and the stability of its expression (Fleming et al., 1994; Langride et al., 1994; Van-Dyke et al., 1996; Ulitzur, 1998; Popova et al., 1999; Unge et al., 1999). The purposes of this work were to study the feasibility of maintaining the genetically modified strain Escherichia coli Z905/pPHL7 in model closed systems based on the brackish lake water, to investigate the varying expression of cloned genes, and to estimate the usability of small closed systems for the solution of similar problems. Adv. Space Res. Vol. 31, No. 7, pp. 1763-1768.2003 Q 2003 COSPAR. Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0273-l 177/03 $30.00 + 0.00

MATERIALS AND METHODS Objects 1‘1~ experiments were made with the strain Escherichiu co/i Z905ipPHL7, produced by transforming the strain E. coli Z905 (hsdR’, hsdM.‘, gal-, met . supE. recA, Tc’), with the recombinant plasmid pPHL7 (Lux’, Ap’). The plasmid contained the genes of the bacterial luminescent system Photobacterium leiognclthi (a- and P-subunits of luciferase and three genes of the fatty acid reductase complex, necessary for aldehyde synthesis), cloned in the vector pUC 18 controlled by &-promoter (Jllarionov and Protopopova, 1985). Introduction of the strain Escherichia coli 2905/pPHL7 into a natural salt medium The natural salt medium was the Lake Shira (Khakasia, Russia) water collected in the central part of the lake, from the surface, in summer. The chemical composition of the water is sulfate-chloride-sodium-magnesium. The water is alkaline (pH=8.9-9.2). The mineral composition of the Lake Shira water is (Zhemchuzhina Khakasii, 1997): so4 Ml99.21

70.72 Cl

21.23

9 W+K)

59.61 Mg

38.43

The water samples were pasteurized three times at a temperature of 70°C for I5 h, at an interval of 24 h. In 24 h after every pasteurization, the water was plated on the peptone-containing agar medium M9 with and without ampicillin in order to test water sterility. Microcosms were created in transparent glass vessels with a total volume of 500 ml, with 250 ml of the medium. The strain E. coli Z905/pPHL7 was plated from a 12-hour culture on the medium M9 with glycerol until the final cell concentration reached 4.105 cells/ml.

Nutrient media Cells of the strain Escherichia coli Z905/pPHL7 were grown on the variants of the liquid medium M9 with different types of added carbon substrate and with or’ without ampicillin. The composition of the peptonecontaining medium M9 was (per 1 ml of distilled water, g): NaH2P04 - 6, K2HP04 - 3, NaCl - 0.5, (NH&SO4 - I, peptone - 5. The medium was autoclaved and under sterile conditions supplemented with 1 ml of 20% MgS04 solution, 1 ml of 0.5% CaCJz solution, 10 ml of 20% glycerol or glucose solution, and, in some experiments ampicillin (50 &ml). The genes of luminescent operon cloned in this strain are subject to glucose-caused catabolic repression. That is why media with and without glucose can be used to estimate changes in the Zux-gene regulation occurring when the microorganism is introduced into salt medium and to study the feasibility of GMMO monitoring by plating samples on the media that contain various carbon substrates. Cultivation Batch cultivation of the strain Escherichia coli Z905/pPHL7 was conducted on an incubator shaker in 30-ml biological test tubes (with 10 ml of the medium) at a temperature of 28’C, at 120 rpm, for 15 hours. The volume of the plated sample from a microcosm was 1 ml. Measurement of optical density The optical density of the bacterial suspension was measured on a KFK photo-electro-calorimeter dish, at a wave length of 540 nm (green filter).

in a 0.3-cm

Measurement of bioluminescence intensity The intensity of bacterial bioluminescence in the liquid culture was measured with a luminometer (made at the Jnstitute of Biophysics SB RAS), with a sensitivity of 10’ quanta/set. The instrument was calibrated by the standard of Hastings and Weber (Hastings and Weber, 1963). Isolation of Esherichia coli Z905/pPHL7 cells The Esherichiu co/i Z905/pPHL7 cells were isolated from the salt medium and from the batch culture by plating the samples on the peptone-containing agar medium M9 with different carbon sources (glucose and glycerol) and with or without ampicillin.

Survival and Alteration of Genetically

3

4

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6

9

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Fig. 1. The luminescence intensity of samples (IO’ quantal(secxml)) taken from the salt medium based on the Lake Shira water, after the introduction of E. co/i Z9OYpPHL7 1000000

100000

T

T

T

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z 3

I-f-I--q

1000

b 100

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Fig. 2. Numerical variations in the Escherichia co/i Z90YpPHL7’population plated on agar nutrient media from the samples of the salt medium based on the Lake Shira water

RESULTS

AND DISCUSSION

To estimate the possibility of long-term survival of the GMMO E. coli 290YpPHL7 under an enhanced mineral salt concentration, we used the mineralized water taken from Lake Shira. After the recombinant strain of E. coli was added to the sterile lake water, parameters of the strain population were measured for 160 days. Bioluminescence in the lake water samples was observed for 9 days after plating (Fig. 1); during that period the population plated on agar media decreased almost tenfold (Fig.2).

Glucose

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Glycerol

0,001 0

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Fig. 3. Specific luminescence intensity (ratio of luminescence intensity (IO’ quanta/(secxml)) to optical density) of samples of the salt medium containing the introduced strain E. co/i 2905IpPHL7 subjected to batch cultivation for 15 h on the media M5 with glucose and M9 with glycerol

Further analysis of the microorganisms’ survival and variations in their characteristics was performed by the method of rapid determination of luminescent strain persi(stence in samples taken from the microcosms. The water samples were plated on the liquid medium M9 that contained peptone and either glycerol or glucose as well as a selective factor (in this case, ampicillin); then batch cultivation was conducted. Batch cultivation of the samples in the presence of ampicillin resulted in the growth of the culture of microorganisms, and luminescence was recorded (Fig 3). The GMMOs introduced into the microcosms based on the Lake Shira water survived through the observation period. The concurrent plating of the samples from the microcosms on solid media showed that the population of the introduced strain, capable of growing on agar media, remained numerically stable - lo4 cells/ml (Fig. 2). A lower level of bioluminescence of the microorganisms plated on the batch culture, observed differences in the sample luminescence intensity in the glucose-containing and the glycerol-containing cultures, and the dynamics of these differences suggest a conclusion about considerable changes in the regulation of luminescent gene expression occurring when the introduced strain adapts itself to new conditions. The glucose repression persists for a month and at the same time the general luminescence level decreases. By the end of the second month, no significant glucose repression is recorded. However, it may still persist, but its manifestation may becomt insignificant because,of the reduction of the bioluminescence intensity by four orders of magnitude relative to the initial level. Yet, the residual bioluminescence of the introduced strain, which is displayed in batch cultures of the samples taken from the microcosms, is sufficient to be detected. Thus, as residual cell concentration in microcosms was 10’ cells/ml, 1 ml taken from a microcosm for analysis contained IO4 cells, and this number of cells was sufficient to be detected.

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Fig. 4. Dynamics of ampicillin resistance of the recombinant strain E. co/i Z905/pPHL7 introduced into the salt medium based on the Lake Shira water Besides estimating the survival of GMMOs in the lake water, we also investigated the heterogeneity of their population in terms of the GMMOs’ resistance to ampicillin. The ability to grow on the antibiotic-containing medium is a marker character indicating the presence of a recombinant plasmid in the cells. The analysis of the GMMO isolate growth on the agar medium containing different ampicillin concentrations showed that the recombinant plasmid was not lost during the experiment (160 days). A reduced threshold of resistance to 50 @ml of ampicillin was recorded in less than 10% of the isolates, irrespective of the extraction time (Fig. 4). Thus, the experiments show that the recombinant strain of E. cofi can exist in the natural salt medium for a long time, considerably reducing the level of cloned gene expression during the first lo-14 days after introduction. The presence of both the cloned lux-genes and the genes of dntibiotic resistance makes the detection of the strain in these conditions much easier, as, under the conditions that are selective for the microorganisms, the luminescent signal can be registered throughout the observation period. ACKNOWLEDGMENTS The work was supported N02-07-06094 and N02-0506250.

by the grants

of Russian

Foundation

for Basic

Research

NOI-05-64615,

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