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Applied and Environmental Microbiology, February 2005, p. 1093-1096, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.1093-1096.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Discovery of glpC, an Organic Solvent Tolerance-Related Gene in Escherichia coli, Using Gene Expression Profiles from DNA Microarrays

Kazunori Shimizu,1 Shuhei Hayashi,1 Takeshi Kako,1 Maiko Suzuki,1 Norihiko Tsukagoshi,2 Noriyuki Doukyu,2 Takeshi Kobayashi,1 and Hiroyuki Honda1*

Department of Biotechnology, School of Engineering, Nagoya University, Chikusa-ku, Nagoya,1 Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan2

Received 21 March 2004/ Accepted 16 September 2004

ABSTRACT

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.

The biological mechanisms of organic solvent tolerance (OST) have been investigated in some microorganisms. In the case of Escherichia coli, it has been reported that MarA, one of the proteins encoded in the mar operon, is a transcriptional activator of mar-sox regulon. acrAB and tolC, the mar-sox regulon genes encoding the efflux pump, contribute to its solvent resistance (6, 7, 14, 15, 18). However, it has been also reported that some genetic determinants seem to give E. coli additional resistance to solvents (1, 3, 4, 13). For example, imp/ostA encoding an 87-kDa minor protein associated with the outer membrane contributes to the n-hexane sensitivity of E. coli. It has also been reported that the solvent resistance of Pseudomonas sp. might be affected by certain cellular components that are not a part of efflux pumps, such as elongation of the O-side chains of lipopolysaccharides or cis-trans isomerization of membrane fatty acids (17).

In our previous study (12), the gene expression profiles of two strains, E. coli K-12 JA300 and its OST spontaneous mutant, OST3410, were used to investigate the biological mechanisms of OST (5). It was reported that marA, the OST-related gene, could be found using only gene expression profiles without any biochemical or biological knowledge.

It seems that the gene expression profiles of plural mutants help us select the genes related to the OST of E. coli. In the present study, we obtained new OST mutants derived from JA300 and compared their gene expression profiles both before and after exposure to organic solvents. Some genes were selected, and their effect on the OST was demonstrated.

At first, we attempted to breed new OST mutants that were derived from E. coli K-12 strain JA300 (5). In accordance with a previous study (5), TK33 and TK31, the spontaneous OST mutants, were screened; these can grow on the LBGMg solid medium (5) overlaid by an organic solvent mixture of cyclohexane and p-xylene (1:1 [vol/vol]) and p-xylene, respectively. The colony morphology of these strains, TK33 and TK31, was not significantly different from that of JA300, their sensitive parent strain, while the growth rates of the mutants were slightly low.

Secondly, the DNA microarray analysis of the three mutants, OST3410, TK33, and TK31, was preliminarily carried out. In comparisons with strain JA300, marA was found to be commonly up-regulated in the mutants (data not shown). However, OST activity cannot be fully explained by the overexpression of marA. OST3408 and OST3410, the OST mutants overexpressing marA, were tolerant to cyclohexane. Our strains, TK33 and TK31, were tolerant to the organic solvent mixture of p-xylene and cyclohexane and to p-xylene, respectively. Therefore, it was strongly suggested that alternative mechanisms for OST activity should be revealed.

Tsukagoshi and Aono have reported that the accumulation of organic solvent inside the cells approached a plateau at 30 min, after the E. coli cells came in contact with an organic solvent (18). Therefore, the DNA microarray analysis was also carried out after exposure to marginal organic solvents for 30 min. DNA microarray analysis was done using a DNA microarray, the IntelliGene E. coli CHIP (Takara Shuzo Co., Ltd., Shiga, Japan), as described previously (12). OST3410, TK33, and TK31 were exposed to cyclohexane, to a solvent mixture of cyclohexane and p-xylene (1:1 [vol/vol]), and to p-xylene, respectively. To collect the expression profiles for each condition, DNA microarray analysis was done three times individually. The median of the intensity data from three DNA microarray analyses was used to represent the intensity of each spot. The genes with an average ratio of more than 2.0 were defined as high-expression genes, and the genes with an average ratio of less than 0.5 were defined as low-expression genes. All spot results were normalized using Excel (Microsoft, Redmond, Wash.) so that the median of all the spot ratios (Cy5/Cy3) was 1.0.

A total of 129 genes in OST3410, 17 genes in TK33, and 11 genes in TK31 were up-regulated; 36 genes in OST3410, 175 genes in TK33, and 103 genes in TK31 were down-regulated. Several genes belonged to the group involving stress response genes. In particular, approximately 30% of the high-expression genes of TK31 and TK33 belonged to the group (data not shown). We focused on the genes commonly up-regulated in three OST mutants and up-regulated in TK33 and TK31. Six genes (576#14, fruA, fruK, glpC, pspA, and pspB) were selected as the candidates (Table 1). Kobayashi et al. have reported that the overexpression of pspA contributes to an improvement in the OST level (13).


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  		TABLE 1. List of genes selected as genes up-regulated by organic solvents

To examine the effect of the selected genes on colony-forming efficiency, a solid-medium assay was done (Fig. 1). Cultures of E. coli (10 ml) were grown in test tubes. When the cell turbidity (optical density at 660 nm) reached 1.0, 4 µl of the diluted cell suspension was spotted on the solid LBGMg medium. Approximately 107, 106, 105, 104, and 103 cells were contained in the spots. The medium was overlaid with an appropriate organic solvent with a thickness of approximately 3 mm and was incubated at 30°C for 30 h. The selected genes were overexpressed using the vector pBluescript II SK(+) (Stratagene, La Jolla, Calif.) according to a previous study (12). To construct the transformants, the gene of interest was amplified with a PCR from the genomic DNA of JA300.



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  		FIG. 1. The colony formation of E. coli on LBGMg agar overlaid with n-hexane. Five clones of JA300 overexpressing the genes selected as those up-regulated by organic solvents were grown for 30 h on LBGMg medium at 30°C. The spots contained approximately 107 (lane 5), 106 (lane 4), 105 (lane 3), 104 (lane 2), and 103 (lane 1) cells.

JA300 cells carrying the high-copy-number glpC plasmid formed many colonies (Fig. 1). It seems that fruA slightly increased the colony formation efficiency of JA300 and that the other three genes did not increase the OST of JA300. It should be noted that the glpC and fruA genes were up-regulated approximately two times in three OST strains (Table 1), while pspA or pspB, i.e., the stress response genes, have been observed to exhibit significantly high expression in OST3410 and TK33 but not in TK31. To confirm the up-regulation of glpC in the three OST mutants, Northern blot analysis was carried out as previously described (12). It seems that the expression of glpC in the three mutants was induced by the marginal organic solvents; as predicted by the results of DNA microarray; in particular, the difference in TK31 results was clearly detected (data not shown).

The sensitivity of deletion mutant of glpC to organic solvents was tested by a solid-medium assay using n-hexane. Unexpectedly, the deletion of glpC did not change the sensitivity to organic solvents. It is likely that the OST was archived by the simultaneous change in expression of many relating genes. Therefore, the decline in OST activity was not so severe even when one of these genes was deleted. On the other hand, when E. coli was transformed by the glpC gene, it was found that the gene expression level remarkably increased and that the level was about 20 times higher than that of control E. coli from the experiment using the DNA microarray. It is likely that OST activity was induced by this dose effect.

There have been no reports to suggest that glpC is one of the mar-sox regulon genes induced by marA (8). Therefore, it was expected that the overexpression of glpC in JA300(pmarA) increased the OST. We constructed the transformant JA300(pglpCmarA). We could not assess the difference between their OST activity on the solid-medium assay using n-hexane, since all three strains, JA300(pglpC), JA300(pmarA), and JA300(pglpCmarA), formed several colonies in all the spots. Hence, we performed the solid-medium assay using cyclohexane, which exhibits greater toxicity to the cells than n-hexane (Fig. 2). JA300(pglpCmarA) formed colonies containing 105 cells (lane 3). On the other hand, JA300(pmarA) formed several colonies (lane 5), containing 107 cells and JA300(pglpC) and JA300(pBS) formed a few colonies (lane 5). It was confirmed that the expression of glpC increased the OST of E. coli even in the transformant overexpressing marA.



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  		FIG. 2. The colony formation of E. coli on LBGMg agar overlaid with cyclohexane at 30°C for 30 h. The spots contained approximately 107 (lane 5), 106 (lane 4), 105 (lane 3), 104 (lane 2), and 103 (lane 1) cells.

Since it has been reported that the OST mutants maintain low intracellular levels of the organic solvents, the amount of solvent entering E. coli cells such as those of JA300(pglpC), JA300(pglpCmarA), JA300(pBS), and JA300(pmarA) was investigated as described previously (18). As shown in Fig. 3, the intracellular n-hexane levels of JA300(pglpC) and JA300(pglpCmarA) were apparently lower than that of JA300(pBS). Further, the level was approximately half for JA300(pglpC) and one-fourth for JA300(pglpCmarA) at 60 min after the strains came in contact with n-hexane. The JA300(pglpCmarA) level was the lowest. Moreover, the intracellular level of JA300(pmarA) was lower than that of JA300(pglpC). These results correspond to the results obtained from the solid-medium assay described above (Fig. 1 and 2).



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  		FIG. 3. Entry of n-hexane into E. coli cells. Each value shown is the mean value for two measurements. Symbols: {circ}, JA300(pBS); {square}, JA300(pglpC); {Delta}, JA300(pmarA); •, JA300(pglpCmarA).

The gene glpC is one of the genes of the glpABC operon encoding the anaerobic glycerol-3-phosphate dehydrogenase. The GlpC subunit functions not as the catalytic subunit but as the membrane anchor for the catalytic GlpAB dimmer and tightly associates with the envelope fraction (10, 11). It has been reported that glycerol and glycerol 3-phosphate dehydrogenase played important roles to acquire osmotolerance in Saccharomyces cerevisiae (9). To determine the effects of glycerol on the OST of E. coli, we investigated colony formation efficiency by a solid-medium assay using modified LBGMg medium which contained 0.1% glycerol substituting for glucose. However, the colony-forming efficiency of JA300 was not changed by the presence of glycerol (data not shown). The overexpression of glpA or glpB did not increase the OST of JA300 (data not shown). Thus, it seems that glycerol, glycerol metabolites, and the specific activity of the anaerobic glycerol-3-phosphate dehydrogenase are not concerned in the OST of JA300.

The possibility may exist that GlpC changes the cell surface properties. It has been reported that overexpression of imp/ostA encoding an 87-kDa minor protein associated with the outer membrane increases the OST of E. coli by reducing the influx of the organic solvent (1). It has also been reported that the cell surface of the OST mutants was less hydrophobic than that of the parent, probably due to an increase in the lipopolysaccharide content (2). The overexpression of glpC encoding a membrane protein might have changed the properties of the cell surface and have resulted in the increase in the OST of E. coli. We carried out preliminary experiments on cell surface hydrophobicity, such as the determination of partition coefficient in a two-phase mixture consisting of p-xylene and aqueous medium. Consequently, it seemed that the hydrophobicity of the cell surface of JA300(pglpC) became lower than that of JA300(pBS) (data not shown).

Moreover, the overexpression of fruA slightly increased the colony formation efficiency of JA300 in Fig. 1. Since FruA, as well as GlpC, is a membrane-associated protein, it is likely that the expression of fruA increases the OST activity of JA300 through a change in the cell surface properties. It also seems that the subclone of fruK did not increase the colony-forming efficiency of JA300; however, it grew faster than the subclones of the other genes. The genes fruA and fruK are located in the same operon, fruBKA (16). Although the mechanism of up-regulation by organic solvents was unclear, this operon may play an important role in the OST of E. coli.

In the future, it will be important to investigate the combined effects of multiple genes on the increase in OST activity. The overexpression of genes encoding transcriptional regulator proteins for which information has been widely reported on the gene-expression network may be one of the effective strategies for increasing the OST of E. coli. For further experiments, we are now planning to collect the time course-gene expression profiles and draw the gene expression network with the use of some effective bioinformatics techniques. It will help us to detect some transcriptional regulator proteins which act as key proteins for the mechanisms of OST in E. coli.

ACKNOWLEDGMENTS

This study was carried out as a part of "The Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers" by the Ministry of Economy, Trade, and Industry (METI) and entrusted by the New Energy and Industrial Technology Development Organization (NEDO). We thank Tomoya Baba in Keio University and Hirotada Mori in Nara Institute of Science and Technology for donating to us the deletion mutant of glpC.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biotechnology, School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Phone: 81 0 52 789 3215. Fax: 81 0 52 789 3214. E-mail: honda{at}nubio.nagoya-u.ac.jp. Back

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Applied and Environmental Microbiology, February 2005, p. 1093-1096, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.1093-1096.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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