JP5532474B2 - Hydrocarbon treatment method - Google Patents

Description

  The present invention relates to a transformed genus Rhodococcus and a method for treating hydrocarbons using the same.

  Rhodococcus (genus Rhodococcus) is a kind of gram-positive bacteria commonly found in soil and the ocean, and a coryneform bacterium with a high G + C content. Rhodococcus bacteria have the ability to decompose and assimilate a number of persistent compounds including petroleum hydrocarbons and polychlorinated biphenyls (PCB), as well as acrylamide and useful enzymes. It is known that there are many producing bacteria such as functional biopolymers including group or extracellular polysaccharides.

  Therefore, it is positioned as an industrially important fungus group, and is expected to be applied to environmental purification and substance production by a bioprocess capable of reducing energy and reducing environmental burden (Non-patent Document 1).

  In particular, when considering a bioprocess, microorganisms that are expected to be applied are required to have properties such as good growth and active metabolic activity in a special environment containing an organic solvent.

  In addition, in order to show good growth while decomposing these microorganisms in the presence of high-concentration non-volatile compounds, microorganisms necessary for purification of the oil-contaminated environment due to an oil spill accident, etc., not only have a resolution for these, but also coexist Therefore, it is required to have high resistance to toxicity of the hardly volatile compound.

  In order to analyze the above-described properties, first, the organic solvent resistance of the microorganism is required as a starting point, and particularly in a bioprocess, growth in the presence of a high concentration organic solvent is required.

  In the research on organic solvent resistance of microorganisms, genetic biochemical research has been conducted mainly on model microorganisms such as Escherichia coli and Pseudomonas bacteria, which are Gram-negative bacteria. A resistance mechanism such as vesicle formation has been proposed (Non-patent Document 2).

  On the other hand, in gram-positive bacteria, genetic chemical research on hydrocarbon degrading genes has progressed, but there are not many studies on organic solvent resistance. This is presumably due to the fact that Gram-positive bacteria are generally considered to have a lower level of organic solvent resistance than negative bacteria.

  However, when considering a bioprocess, information on resistance to organic solvents under conditions close to the actual use environment is required for microorganisms at a very close to application stage. As described above, since Rhodococcus bacteria are expected to be applied to bioprocesses, it is necessary to accumulate knowledge about the organic solvent resistance of the bacteria.

  Iwabuchi et al. Found that Rhodococcus rhodochrous S-2 strain is a high-concentration oil-resistant oil-degrading bacterium, and as a result of investigating its oil resistance, the extracellular polysaccharide produced by the bacterium (hereinafter, It was revealed that "EPS" is deeply involved in the resistance of hardly volatile organic solvents such as long-chain alkanes.

  Furthermore, when the colony morphology and solvent resistance of Rhodococcus genus bacteria were examined, rough type bacteria with a low EPS production amount have high affinity for the solvent and as a result are solvent-sensitive. On the other hand, the EPS production amount is large. Since mucoid bacteria showed resistance, it was clarified that there is a high correlation between colony morphology and organic solvent resistance in bacteria belonging to the same genus.

  EPS has also been shown to impart solvent resistance to solvent-sensitive rough bacteria, and from these, the formation of mucoid colonies is one index for considering the solvent resistance of the genus bacteria. (Non-Patent Document 3).

  Rhodococcus erythropolis PR4 strain is a strain isolated as a degrading bacterium of pristane (2,6,10,14-tetramethyl-pentadecane) which is a kind of branched alkane (Non-patent Document 4). It is a strain that changes its colony form based on the production of EPS over time from rough type to mucoid type to rough type.

  Since this strain is known to exhibit resistance to a hardly volatile organic solvent, it has been selected as a genome analysis strain and has also begun to develop a host-vector system. Therefore, the Rhodococcus erythropolis PR4 strain was expected to become a strain with a genetically engineered system among the bacteria of the genus Rhodococcus in the near future. Since it is a strain having high industrial utility value, genome analysis was performed and the entire sequence of the plasmid was published (Non-patent Document 5). The applicant also revealed the chemical structure of two types of EPS (Non-patent Document 6).

  In research on resistance to organic solvents in Rhodococcus bacteria and related bacteria, changes in the ratio of saturated and unsaturated fatty acids in the cytoplasmic membrane and the ratio of cis / trans fatty acids under various stress conditions such as temperature changes and nutrient deficiencies Although it is known that a change in membrane structure causes a change in the permeability of the membrane and adapts to the environment, this change in permeability of the membrane is said to contribute to the resistance to organic solvents (Non-Patent Documents 7 and 8). ).

  Loredana et al. Also analyzed R. eythropolis 20S-E1-c strain, which is the same kind as PR4 strain, in a study analyzing the interaction between hydrocarbons and various strains. However, it was reported that other Acinetobactor genus, Rhizomonas genus, and Pseudomonas genus bacteria were adsorbed to C16 and no metastasis was confirmed (Non-patent Document 9). ).

  In addition, Iwatsuki et al. Revealed that Rhodococcus erythropolis PR4 strain was transferred to tetradecane, pentadecane, hexadecane, pristane, squalane, etc. having 14 or more carbon atoms, and these hydrocarbons could be metabolized and decomposed ( Patent Document 1).

JP 2006-325433 A

Finnerty, W. R. et al. (1992) Annual Review of Microbiology, p193-218. Ramos, J. L. et al. (2002) Annual Review of Microbiology, p743-768. Iwabuchi, N. et al. (2000) Applied Environmental Microbiology, 66: 5073-5077. Komukai-Nakamura, S. et al. (1996) Journal of Fermentation and Bioengineering, 82: p570-574 (Sekine et al. 2006, Appl.Environ.Microbiol, 8: 334-346) Urai, M. et al. (2006) Carbohydr Res, 341: 616-623,766-775 Cronan, J.E et al. (2002) Microbiol, 5: 202-205 Whyte, L. G. et al. (1999) Appl. Environ. Microbiol, 65: 2961-2968 Loredana, S. Et al. (2004) Appl.Environ.Microbiol, 70: 6333-6336

  As described above, although conventional Rhodococcus microorganisms are useful for the decomposition and metabolism of hydrocarbons, there are restrictions on the hydrocarbons that can be decomposed and metabolized, such as being unable to transfer to hydrocarbons with a certain number of carbon atoms or less. There was a problem.

  Moreover, since conventional Rhodococcus microorganisms cannot grow in the presence of hydrocarbons having a low carbon number, they could not be used for the decomposition and metabolism of such hydrocarbons.

  Therefore, the present invention improves the viability of Rhodococcus bacteria against low carbon number hydrocarbons by providing a transformant of Rhodococcus, and also decomposes / decomposes hydrocarbons with fewer carbon numbers than before. The object is to provide an effective method for metabolism.

As described above, Rhodococcus bacteria are presumed to have a characteristic organic solvent resistance mechanism different from other microorganisms, but the details are still unclear.
Under such circumstances, the inventor of the present application (1) Rhodococcus erythropolis PR4 strain (hereinafter, Rhodococcus erythropolis is abbreviated as “Rh”, Rhodococcus erythropolis PR4 strain is referred to as “PR4”. May be abbreviated as “.” Because of the fact that genome analysis has been carried out and (2) the interaction with the organic solvent exhibited by the strain is extremely characteristic. The material has been studied from the viewpoint of genetic biochemistry and physicochemistry, considering it as a suitable material for research.

  As a result of analyzing the physicochemical interaction between PR4 and alkanes so far, for example, the addition of pristane increases the lipophilicity of the cell surface, and as a result, the interface Gibbs energy at the medium / alkane interface decreases. It became clear that the cells can metastasize into the alkane.

  Therefore, in order to clarify the cause of these differences in physicochemical properties at the molecular level, the present invention further includes (1) examination of related proteins by proteome analysis, and (2) mutation introduction using transposon. Related genes were examined.

  PR4 is an alkane-degrading bacterium isolated as a pristane (2,6,10,14-tetramethylpentadecane) -degrading bacterium, but the interaction with the alkane varies depending on the length of the carbon number of the alkane. This property of the bacterium is very characteristic, and its application to green biotechnology has been proposed and genome analysis has already been performed.

  Therefore, in the present invention, proteins related to the interaction with alkanes were examined by proteome analysis, and total proteins were extracted from PR4 grown under various culture conditions and analyzed by LC-MS / MS.

  As a result, the protein GroEL2, which was highly expressed with high reproducibility by the addition of pristane, was found. Subsequently, when the groEL2 gene was cloned and introduced into various strains, the localization of the cells was changed, and it became possible to grow under conditions that could not be grown in the parent strain.

  In addition, it was found that the growth was increased about 100 times compared to the parent strain under the dodecane addition condition. From the above, it was found that GroEL2 is involved in the determination of cell localization and growth in a bilayer culture system. That is, the Rhodococcus erythropolis PR4 strain into which the groEL2 gene has been introduced can grow in the presence of C6 to C8 and cannot grow in the parent strain. For example, even if C8 is added to the medium, It became able to grow by adsorption.

  The present invention provides a transformant in which the GroEL2 gene is introduced into the genus Rhodococcus erythropolis, and this as a means for decomposing and metabolizing hydrocarbons. Since the transformant of Rhodococcus erythropolis into which GroEL2 gene has been introduced can grow even in the presence of hydrocarbons of C14 or lower and C6 or higher, it has the potential to decompose and metabolize these hydrocarbons. Since C10-C14 hydrocarbons, which could not be decomposed and metabolized in the converted form, have a higher transfer than adsorption, they can be decomposed and metabolized.

  According to the present invention, by providing a novel transformant of Rhodococcus bacteria, the viability of Rhodococcus bacteria with respect to low carbon number hydrocarbons is improved, and hydrocarbons having a smaller number of carbon atoms than conventional ones are also provided. It is possible to provide an effective method for decomposing and metabolizing.

It is the figure which showed notionally the series of processes from the start of a hydrocarbon process to a substance production. 1 is a conceptual diagram of a hydrocarbon treatment system 2. FIG. This is an SDS-PAGE pattern of PR-4 strain. It is the band pattern of the PCR product which amplified groEL2 using the primer 366F-11 and GroEL-R1. Lanes 1 to 5 show PCR products obtained by amplifying groEL2 using primers 366F-11 and GroEL-R1. It is a band pattern related to migration confirmation of a PCR product obtained by TA cloning. Lane 1 is a groEL2 PCR product, lane 2 is a topovector, lane 3 is a sample obtained by EcoRI treatment, lane 4 is a topovector + groEL2, and lane 5 is a sample obtained by EcoRI treatment. The preparation of the plasmid is shown, and (A) shows the migration results of insert check and orientation confirmation. Lane 1 is a sample in which topovector + groEL2 is treated with EcoRI, lane 2 is a sample in which pk4 + groEL2 is treated with EcoRI, lane 4 is a sample in which pk4 + groEL is treated with ApaI and KpnI. (B) is a schematic diagram of pk4 + groEL. It is a microscope picture which shows the dynamics in each growth condition of PR-4 (pk4 + groEL2), (A) shows C8 addition, (B) shows C12 addition, (C) shows C19 addition. This shows the interaction between PR-4 (pK4 + groEL2) and alkane. It is a microscope picture which shows the dynamics in C19 addition conditions of PR-4 (pK4 + groEL2), (A) is PR-4, (B) is PR-4 (pK4 + groEL2). It is a microscope picture which shows the dynamics in C12 addition conditions of PR-4 (pK4 + groEL2), (A) is PR-4, (B) PR-4 (pK4 + groEL2). It is a microscope picture which shows the dynamics in C8 addition conditions of PR-4 (pK4 + groEL2), (A) is PR-4, (B) is PR-4 (pK4 + groEL2). PR-4 (pK4 + groEL2) is a characteristic diagram showing the number of viable bacteria in the presence of C12, ○ is the number of viable bacteria in the wild-type nutrient medium only, and △ is for the wild-type strain with C19 added □ is for wild-type C12 addition conditions, and ◇ is for PR-4 (pK4 + groEL2) C12 addition conditions. Photomicrograph showing introduction of groEL2 gene into other species (R-1 strain). Photomicrograph showing introduction of groEL2 gene into other species (R-2 strain). Photomicrograph showing introduction of groEL2 gene into other species (S-1 strain). Photomicrograph showing introduction of groEL2 gene into other species (R-2 strain). It is a characteristic view which shows interaction with PR-4 (pK4 + groEL2) and alkane. A photomicrograph showing the interaction between PR-4 (pK4 + groEL2) and alkane in an environment where magnesium chloride is substantially absent from the IB medium. It is a characteristic view which shows the interaction of PR-4 (pK4 + groEL2) and alkane in the environment where magnesium chloride does not substantially exist in IB culture medium.

  Next, embodiments of the present invention will be described in more detail. The “hydrocarbon” includes chain hydrocarbons and cyclic hydrocarbons.

  “Hydrocarbon treatment” means that a transformant of the genus Rhodococcus erythropolis, particularly a hydrocarbon that can be decomposed and metabolized by the PR4 strain, is added to the medium. “Transition into an organic solvent” means that the Rhodococcus erythropolis PR4 strain existing in the aqueous solvent is adsorbed or transferred from the aqueous solvent to the carbon hydrocarbon contained in the organic solvent.

  Rhodococcus erythropolis PR4 strain can take either a rough type or a mucoid type according to the progress of culture. Either form can be transferred into an organic solvent.

  As an aqueous solvent containing a culture medium component, a general bacterial culture medium can be used. Moreover, other culture medium components may be included in the culture medium for general bacteria. Examples of the medium for general bacteria include IB liquid medium, YG liquid medium, LB medium, marine broth, nutritive broth, tryptic soy broth and the like. Among general bacterial media, it is particularly preferable to use an IB liquid medium.

  When the IB liquid medium is used, yeast extract is an important medium component when Rhodococcus erythropolis PR4 strain is transferred from an aqueous solvent to an organic solvent. Even when the Rhodococcus erythropolis PR4 strain is added to an aqueous solvent in which no yeast extract is present, the transfer to an organic solvent hardly occurs.

  The same applies to inorganic salts. Specifically, the concentration of magnesium salt, more specifically, the concentration of magnesium chloride salt in the medium is preferably limited. Preferably, the magnesium salt, particularly, the magnesium chloride salt is substantially not contained. The localization of the transformant to hydrocarbons, ie the transfer of transformants from aqueous medium to hydrocarbons, and the transfer of hydrocarbons into hydrocarbons over adsorption rather than adsorption. Is important.

  The addition amount of the yeast extract in the aqueous solvent is preferably 0.05% (w / w) or more, based on the total amount of the aqueous solvent (100%), 0.5 to 5% (W / w) is more preferable, and about 1% (w / w) is even more preferable.

  The transformed Rhodococcus genus bacteria can grow on C14 or less hydrocarbons, preferably hydrocarbons having 6 or more carbon atoms, that could not be metabolized by PR4 conventionally, and at least these hydrocarbons Because it can be adsorbed, these low-carbon hydrocarbons can be metabolized and decomposed.

  The upper limit of the carbon number is not limited. For example, even if it is solid at room temperature, it is only necessary that the solid hydrocarbon can be dissolved into a liquid by coexistence with other hydrocarbons.

  The proportion of hydrocarbon in the organic solvent applied to the transformant is not particularly limited, but is preferably 20% (v / v) or more, more preferably 40% (v / v) or more, and 60% (v / V) is more preferred. You may mix 1 type, or 2 or more types of hydrocarbons.

  FIG. 1 is a diagram conceptually showing a series of processes from the start of hydrocarbon processing to the production of substances. FIG. 1 (a) is a view showing a state immediately after the Rhodococcus erythropolis PR4 strain (parent strain and transformant) 12 is added to the aqueous solvent 10. Immediately after the addition of Rhodococcus erythropolis PR4 strain 12, the Rhodococcus erythropolis PR4 strain 12 exists in a state of being dispersed around the organic solvent 14, that is, in the aqueous solvent 10.

  Thereafter, as shown in FIG. 1 (b), Rhodococcus erythropolis PR4 strain 12 moves into the organic solvent 14 and agglomerates in the organic solvent 14. At this point, Rhodococcus erythropolis PR4 strain 12 is hardly present in aqueous solvent 10.

  As shown in FIG. 1C, Rhodococcus erythropolis PR4 strain 12 metabolizes and grows hydrocarbons in organic solvent 14 and produces water-soluble product P. Since the organic solvent 14 is hydrophobic, the water-soluble product P moves from the organic solvent 14 into the aqueous solvent 10 and is dispersed in the aqueous solvent 10.

  Hydrocarbon treatment may or may not be carried out, but from the viewpoint of bringing more Rhodococcus erythropolis PR4 strain 12 into contact with organic solvent 14 and ensuring the amount of dissolved oxygen in the medium. It is preferable to perform the treatment while stirring. Stirring can be performed by using, for example, a stirring device or a shaking device.

  FIG. 2 is a conceptual diagram of the hydrocarbon treatment system 2. As shown in FIG. 2, the hydrocarbon treatment system 2 includes an organic solvent supply unit 20, an aqueous solvent supply unit 22, a fungus body addition unit 24, a treatment unit 26, and a product separation unit 28. Yes.

  The organic solvent supply means 20 supplies an organic solvent containing hydrocarbons to the processing means 26 described later.

  The aqueous solvent supply means 22 supplies an aqueous solvent containing a medium component to the processing means 26 described later. As an aqueous solvent containing a culture medium component, a general bacterial culture medium can be used.

  Moreover, other culture medium components may be included in the culture medium for general bacteria. Examples of the medium for general bacteria include IB liquid medium, YG liquid medium, LB medium, marine broth, nutritive broth, tryptic soy broth and the like. Among general bacterial media, it is particularly preferable to use an IB liquid medium.

  The fungus body adding means 24 adds the Rhodococcus erythropolis PR4 strain to the processing means 26 described later. The Rhodococcus erythropolis PR4 strain can take either a rough type or a mucoid type according to the progress of the culture, but any form can be used.

  The microbial cell addition means 24 can also be provided with a pre-culture means having a function of pre-culturing Rhodococcus erythropolis PR4 strain in advance. Pre-culture can be performed, for example, by shaking culture of Rhodococcus erythropolis PR4 at 28-30 ° C. using the IB medium.

  The treatment means 26 treats the hydrocarbons contained in the organic solvent by Rhodococcus erythropolis PR4 in a medium composed of an organic solvent and an aqueous solvent. The organic solvent is supplied from the organic solvent supply means 20, and the aqueous solvent is supplied from the aqueous solvent supply means 22.

  As a result, an organic solvent-aqueous solvent bilayer culture system is formed. Then, the Rhodococcus erythropolis PR4 strain is supplied from the cell supply means 24 into the aqueous solvent. Thereafter, the organic solvent and the aqueous solvent are stirred by a stirrer (not shown), and Rhodococcus erythropolis PR4 strain starts hydrocarbon metabolism and substance production. Hydrocarbon treatment conditions are performed, for example, at a temperature of 28 to 30 ° C. for several days to two weeks while stirring the medium with a stirring device (not shown). In addition, process conditions can be suitably set according to the kind of hydrocarbon to process.

  The product separation means 18 separates the product P, the organic solvent 12 and the aqueous solvent 10 produced in the aqueous solvent. As the separation method, a conventionally known separation / purification method (various chromatography, various electrophoresis or the like) can be used. In addition, using the property of the organic solvent-aqueous solvent two-layer culture system, the medium is allowed to stand for a predetermined time, and after the organic solvent and the aqueous solvent are separated into two layers, only the aqueous solvent is separated. The product P can also be purified.

  In addition, since Rhodococcus erythropolis PR4 strain | stump | stock exists in an organic solvent, isolation | separation of an aqueous solvent and a microbe is also easy. After the treatment, the product separation means 18 separates and purifies the organic solvent 12, the aqueous solvent 10 and the product P.

  Rhodococcus erythropolis PR4 strain is listed as NBRC10087 in the catalog issued by the Biological Resource Information Division, National Institute of Product Evaluation and Technology, and could be freely distributed before filing.

Next, embodiments of the present invention will be described in more detail.
[Example 1] Treatment of various hydrocarbons (1) Preparation of IB liquid medium An IB liquid medium was prepared in the following manner. That is, 1 L of Milli-Q was prepared, 10 g of glucose (Wako Pure Chemical Industries), 10 g of yeast extract (DIFCO LABORATOREIS), 0.2 g of MgCl ₂ .7H ₂ O (Wako Pure Chemical Industries), CaCl ₂・ 0.1 g of 2H ₂ O (Wako Pure Chemical Industries), 0.1 g of NaCl (Wako Pure Chemical Industries), 0.02 g of FeCl ₂ .6H ₂ O (Wako Pure Chemical Industries), (NH ₄ ) ₂ SO ₄ (Wako Pure Chemical Industries, Ltd.) 0.5 g was added, adjusted to pH 7.2 with NaOH solution, and then autoclaved at 121 ° C. for 15 minutes.

(2) Various hydrocarbons As n-alkanes, n-hexane (C6), n-octane (C8), n-nonane (C9), n-decane (C10), n-undecane (C11), n-dodecane ( C12), n-tridecane (C13), n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), and n-octadecane (C18) were used. Pristane (C19) and squalane (C30) were used as branched alkanes.

(3) Test strain As the test strain, Rhodococcus erythropolis PR4 strain and Rhodococcus rhodochrous S-2 strain were used.

(4) Hydrocarbon treatment method (treatment using parent strain)
The test strain shown in (3) above was inoculated with a platinum loop into the IB liquid medium prepared in (1) above, and cultured with shaking at 28 ° C. for 3 days. 1 ml of the preculture was collected and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The obtained precipitate was suspended by adding 1 ml of physiological saline, and centrifuged again. Thereafter, this washing operation was repeated twice, and the resulting precipitate was suspended in 1 ml of physiological saline, which was used as a stock solution.

The stock solution was appropriately diluted so that the initial concentration of the test strain was 10 ⁴ cfu / ml, and added to fresh IB medium contained in a φ24 test tube (IWAKI GLASS). Subsequently, the various hydrocarbons shown in (2) above were added to a final concentration of 5% (v / v), followed by shaking culture at 28 ° C. and 110 rpm. Then, on the third day from the start of culture, the growth status of the test strain and the location where the test strain was localized were observed. Here, when it was present in the organic solvent, it was “internal”, and when it was present on the surface of the organic solvent, it was “superficial”. The results are shown in Table 1.

  When Rhodococcus erythropolis PR4 was used, the state of the culture solution was compared. As a result, an increase in turbidity in an organic solvent and an aqueous solvent was confirmed under the condition that an n-alkane having 14 or more carbon atoms and a branched alkane were added. It was done.

  Further, when microscopic observation was performed, it was confirmed that Rhodococcus erythropolis PR4 strain was transferred and present in hydrocarbon particles having 14 or more carbon atoms.

  Under the conditions in which n-alkane having 10 to 12 carbon atoms was added, an increase in turbidity was observed only at the interface between the organic solvent and the aqueous solvent. When these were observed under a microscope, it was confirmed that Rhodococcus erythropolis PR4 strain was adsorbed on the surface of the hydrocarbon particles.

  Under the condition using n-alkane having 8 or less carbon atoms, almost no turbidity was observed. Further, when microscopic observation was performed, since Rhodococcus erythropolis PR4 strain was not observed in the aqueous solvent and organic solvent, it is expected that the number of Rhodococcus erythropolis PR4 strain is significantly smaller than other conditions. It was.

  On the other hand, the same examination was performed for Rhodococcus rhodochrous S-2 strain (in the table, referred to as “S-2 strain”). As a result, the formation of an emulsion was observed in the whole culture solution under the conditions in which an n-alkane having 14 or more carbon atoms and a branched alkane were added. Further, when microscopic observation was performed, it was observed that Rhodococcus rhodochrous S-2 was present in an aqueous solvent under these conditions, and that Rhodococcus rhodochrous S-2 was adsorbed on the surface of the hydrocarbon particles. It was.

  Under the condition where n-alkane having 12 or less carbon atoms was added, formation of an emulsion was not observed, and Rhodococcus rhodochrous S-2 strain could not be confirmed in the culture solution.

  From this, it was found that Rhodococcus erythropolis PR4 strain and Rhodococcus rhodochrous S-2 strain have different interactions with hydrocarbons. It was also suggested that Rhodococcus erythropolis PR4 recognizes hydrocarbons with different carbon numbers and interacts with them according to the carbon number.

[Example 2] Examination of influence of mixing ratio of different hydrocarbons on hydrocarbon treatment (when parent strain is used)
Two types of hydrocarbons with different interactions were selected, changed in their proportions, added to the IB liquid medium, and the effect on the treatment of hydrocarbons by Rhodococcus erythropolis PR4 was examined.

  One platinum loop of Rhodococcus erythropolis PR4 was inoculated into IB liquid medium, and cultured with shaking at 28 ° C. for 3 days. 1 ml of the preculture was collected and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The obtained precipitate was suspended by adding 1 ml of physiological saline, and centrifuged again. Thereafter, this washing operation was repeated twice, and the resulting precipitate was suspended in 1 ml of physiological saline, which was used as a stock solution.

The stock solution was appropriately diluted so that the initial concentration of Rhodococcus erythropolis PR4 was 10 ⁴ cfu / ml, and Rhodococcus erythropolis PR4 was added to the separately prepared IB medium. Subsequently, different hydrocarbons were added, respectively, and cultured with shaking at 28 ° C. and 110 rpm.

  Hydrocarbons include pristane (C19), which is a hydrocarbon that Rhodococcus erythropolis PR4 can migrate and metabolize, and hydrocarbon that Rhodococcus erythropolis PR4 absorbs and metabolizes on the surface of organic solvents. A mixture of some dodecane (C12) was used. And each mixing ratio was changed so that the final concentration of the mixed hydrocarbon might be 5% (v / v), and it added to the culture solution, and processed the hydrocarbon.

  Also, hydrocarbons were treated in the same manner as described above using pristane and octane (C8), which is a hydrocarbon that Rhodococcus erythropolis PR4 strain cannot migrate and grow. The results are shown in Table 2.

  As shown in Table 2, when dodecane and pristane were mixed, the transfer of Rhodococcus erythropolis PR4 to an organic solvent was confirmed when the mixing ratio of dodecane and pristane was 8: 2 to 1: 9.

  On the other hand, when pristane and octane were mixed, the transfer of Rhodococcus erythropolis PR4 to an organic solvent was confirmed when the mixing ratio of octane and pristane was 6: 4 to 1: 9.

[Example 3] Examination of adaptation of Rhodococcus erythropolis PR4 strain (using parent strain)
Rhodococcus erythropolis PR4 strain (referred to as “PR4 strain” in the table) was inoculated into IB liquid medium in a platinum loop, and cultured with shaking at 28 ° C. for 3 days. 1 ml of the preculture was collected and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The obtained precipitate was suspended by adding 1 ml of physiological saline, and centrifuged again. Thereafter, this washing operation was repeated twice, and the resulting precipitate was suspended in 1 ml of physiological saline, which was used as a stock solution.

The stock solution was appropriately diluted so that the initial concentration of Rhodococcus erythropolis PR4 strain was 10 ⁴ cfu / ml and added to a fresh IB liquid medium contained in a φ24 test tube (IWAKI GLASS). Subsequently, the final concentration of pristane (C19) was added to 5% (v / v), followed by shaking culture at 28 ° C. and 110 rpm for 3 days.

After 3 days from the start of culture, Rhodococcus erythropolis PR4 cultured in IB liquid medium supplemented with pristane (C19) was recovered. The recovered Rhodococcus erythropolis PR4 strain was added to a separately prepared IB liquid medium so that the initial concentration was 10 ⁴ cfu / ml. This time, the final concentration of dodecane (C12) was added to 5% (v / v), followed by shaking culture at 28 ° C. and 110 rpm.

  As a control, Rhodococcus erythropolis PR4 strain was cultured in an IB liquid medium supplemented with dodecane (C12), and then Rhodococcus erythropolis PR4 strain was cultured in an IB liquid medium supplemented with pristane (C19).

  On the third day from the start of the culture, the growth status of Rhodococcus erythropolis PR4 strain and the location where Rhodococcus erythropolis PR4 strain was localized were observed. Here, when the Rhodococcus erythropolis PR4 strain is present in the hydrocarbon, it is “internal”, and when it is present on the surface of the hydrocarbon, it is “superficial”. The results are shown in Table 3.

  As shown in Table 3, it was found that Rhodococcus erythropolis PR4 strain was transferred into dodecane (C12) in the same manner as when pristane (C19) was treated alone. Conversely, Rhodococcus erythropolis PR4 strain cultured in a medium supplemented with dodecane (C12) was recovered and cultured in a medium supplemented with pristane (C19). Rhodococcus erythropolis PR4 strain was found in pristane (C19). It did not migrate and was found to be present on the surface of pristane (C19). Therefore, during the first stage treatment, the cell wall and / or cell membrane of Rhodococcus erythropolis PR4 changes according to the type of hydrocarbon (adaptation), and its properties are It was inferred that the hydrocarbon used in the treatment could be maintained even if it changed.

Example 4
Next, related proteins by proteome analysis and related genes by mutagenesis using transposon will be described.

Culture medium
IB agar medium: glucose of milli-Q water 800 ml to 8 g (Wako Pure Chemical Industries, Ltd.), yeast the Extract of 8 g (Becton, Dickison and company ), 0.16 g MgCl 2 · 7H 2 O of (Wako Pure Chemical Industries), 0.08 g CaCl ₂・ 2H ₂ O (Wako Pure Chemical Industries), 0.08 g NaCl (Wako Pure Chemical Industries), 0.016 g FeCl ₂・ 6H ₂ O (Wako Pure Chemical Industries), 0.4 g (NH ₄ ) ₂ SO ₄ (Wako Pure Chemical Industries) was added to adjust to pH 7.2, 12 g of agar (Wako Pure Chemical Industries) was added, and autoclaved at 121 ° C. for 15 minutes.

  Luria-Bertani (LB) agar medium: Add 8 g NaCl (Wako Pure Chemical Industries), 8 g trypton (Difco), 4 g yeast extract (Becton, Dickison and company) to 800 ml Milli-Q water, pH After adjusting to 7.2, 12 g of agar (Wako Pure Chemical Industries) was added, and autoclaved at 121 ° C. for 15 minutes.

  Luria-Bertani (LB) liquid medium: Add 8 g NaCl (Wako Pure Chemical Industries), 8 g trypton (Difco), 4 g yeast extract (Becton, Dickison and company) to 800 ml Milli-Q water, pH After adjusting to 7.2, the mixture was autoclaved at 121 ° C. for 15 minutes.

reagent
0.5M EDTA: While mixing MilliQ water in a beaker, 93.05 g of EDTA · 2Na (DOJINDO) was added while stirring with a stirrer, 10 g of NaOH was added, pH was adjusted to 8.0 with 3 N NaOH, and autoclaved.

  10% SDS: 500 ml of milli-Q water was placed in a medium bottle, and 14.42 g of SDS (Wako Pure Chemical Industries, Ltd.) was added and shaken to dissolve. Waiting for the foam to disappear, adjusted to pH 7.2 with NaOH and stored at room temperature.

  1 M tris HCl: 121.1 g of TRIZMA base (SIGMA) was dissolved in Milli-Q water, adjusted to pH 8.0 with HCl (Wako Pure Chemical Industries), and then filled up to 1000 ml with Milli-Q water.

  0.5 M tris-HCl: 6 g of TRIZMA base (SIGMA) was dissolved in Milli-Q water, adjusted to pH 6.8 with HCl (Wako Pure Chemical Industries), and then filled up to 100 ml with Milli-Q water.

  50 mM tris-HCl: 0.605 g of TRIZMA base (SIGMA) was dissolved in milliQ water, adjusted to pH 8.0 with HCl (Wako Pure Chemical Industries), and then filled up to 100 ml with milliQ water.

  Neutral phenol: 1000 ml of 1 M tris HCl was autoclaved and allowed to cool to room temperature. Next, phenol (Wako Pure Chemical Industries, Ltd.) was placed in a 68 ° C warm bath to dissolve the crystals and make them liquid. To this phenol, 8-hydroxyquinoline (Wako Pure Chemical Industries, Ltd.) as an antioxidant was added to a final concentration of 0.1%, stirred, allowed to stand to separate into two layers, and then the upper layer was removed. This was repeated to adjust the phenol to pH 8.0.

  3 M sodium acetate: Milli-Q water was added to a beaker, and 204.05 g of sodium acetate (Wako Pure Chemical Industries, Ltd.) was added while mixing with a stirrer. Glacial acetic acid was added thereto to adjust to pH 5.2. Filled up to 500 ml with MilliQ water and autoclaved.

  Solution I: While adding Milli-Q water to a beaker and mixing with a stirrer, 15 ml of 1 M glucose (Wako Pure Chemical Industries), 7.5 ml of 1 M Tris HCl (pH 8.0), 6 ml of 0.5 M EDTA (pH 8.0) In addition, it was filled up to 300 ml and autoclaved.

  Solution II: 50 ml of Milli-Q water was added to the Falcon tube, then 0.4 g of NaOH (Wako Pure Chemical Industries) and 5 ml of SDS (Wako Pure Chemical Industries) were added and shaken and mixed.

  TE buffer: 10 ml of 1 M tris HCl (pH 8.0) and 2 ml of 0.5 M EDTA (pH 8.0) were added to Milli-Q water and filled up to 1000 ml with Milli-Q water. Next, this was autoclaved and allowed to cool to room temperature.

  50 × TAE buffer: 242 g of TRIZMA base (SIGMA), 57.1 ml of acetic acid and 100 ml of 0.5M EDTA were added to Milli-Q water and filled up to 1000 ml, followed by autoclaving.

  1 × TAE buffer: 20 ml of 50 × TAE buffer was added to Milli-Q water and filled up to 1000 ml with Milli-Q water.

  Proteinase K: A product of Promega Corporation was used. The solution was diluted to a concentration of 2 mg / ml using Milli-Q water, and sterilized by filtration using 0.22 μm mirex.

  IPTG: TaKaRa product was used. 0.238 g of IPTG was dissolved in 10 ml of Milli-Q water, and sterilized by filtration using 0.22 μm Millex (Millipore).

  Xgal: TaKaRa product was used. Dilute to a concentration of 20 mg / ml with MilliQ water and filter sterilize using 0.22 μm Millex (Millipore).

  Km: Wako Pure Chemical Industries product was used. Dilution was performed to a concentration of 100 mg / ml using Milli Q water, and sterilization by filtration was performed using 0.22 μm Millex (Millipore).

  Amp: Wako Pure Chemical Industries product was used. Dilution was performed to a concentration of 100 mg / ml using Milli Q water, and sterilization by filtration was performed using 0.22 μm Millex (Millipore).

  Ts: SIGMA product was used. Dilution was performed to a concentration of 100 mg / ml using Milli Q water, and sterilization by filtration was performed using 0.22 μm Millex (Millipore).

  Denaturing buffer: SDS (Wako Pure Chemical Industries) 0.1 g was dissolved in 50 mM tris-HCl (pH 8.0), and Milli-Q water was filled up to 100 ml.

Working Reagent (coloring reagent): First, the necessary amounts of Working Reagent A and Working Reagent B included in the BCA ^™ Protein Assay Kit were prepared at a ratio of 50: 1.

  20% (W / V) SDS: 1 g of sodium dodecyl sulfate (Wako Pure Chemical Industries) was dissolved in 4 ml of milli-Q water.

  sample buffer: 1.25 ml of 0.5 M tris-HCl, 2.5 ml of glycerol (Wako Pure Chemical Industries), 1.9 ml of 20% SDS, and 0.47 ml of β-mercaptoethanol (SIGMA) were added to 3.18 ml of milli-Q water.

  2 x sample buffer: 0.2 ml of milli-Q water, 1.25 ml of 0.5M tris-HCl, 5 ml of glycerol (Wako Pure Chemical Industries), 1.9 ml of 20% SDS, 0.2 ml of 1% bromophenol blue, β-mercaptoethanol ( 0.95 ml of SIGMA) was added.

  Sample buffer for molecular weight marker: Milli-Q water 3.55 ml, 0.5 M tris-HCl 1.25 ml, glycerol (Wako Pure Chemical Industries) 2.5 ml, 10% SDS 2 ml, 0.5% bromophenol blue 0.2 ml, β-mercaptoethanol 0.5 ml of (SIGMA) was added.

  Molecular weight marker for SDS-PAGE: 5 μl of molecular weight marker and 95 μl of sample buffer for molecular weight marker were added and mixed.

  running buffer: Milli-Q water in 500 ml, tris aminomethane nuclease and protease tested (Nacalai Tesque Co., Ltd.) 15.15 g, glycine (Wako Pure Chemical Industries) 72 g, SDS 5 g, 100 ml . 100 ml of this and 900 ml of milli Q water were added and mixed.

  Cleaning liquid: 100 ml of methanol (Wako Pure Chemical Industries) was mixed with 100 ml of Milli-Q water and refrigerated.

  Stopping solution: 100 ml of acetic acid (Wako Pure Chemical Industries) was mixed with 190 ml of Milli-Q water and refrigerated.

  Sensitizer: 10 mg of sodium tThiosufate (Wako Pure Chemical Industries, Ltd.) was added to 50 ml of Milli-Q water, mixed and refrigerated.

  Silver nitrate aqueous solution: 50 mg of silver nitrate (SIGMA) was added to 50 ml of milli-Q water and mixed, and the mixture was refrigerated with light shielding.

  Developer: 1 g of sodium carbonate (Wako Pure Chemical Industries) was added to 50 ml of milli-Q water. Immediately before use, 50 μl of formaldehyde sodium (nacalai tesque) was added.

  Sodium thiosufate (Wako Pure Chemical Industries, Ltd.) 791 mg was added to 100 mM sodium thiosulfate: Milli-Q water (50 ml), mixed, and refrigerated in the dark.

  1 M DDT: 771 mg of ditio threitol (Wako Pure Chemical Industries) was added to 5 ml of Milli-Q water and mixed.

  1 M ammonium bicarbonate: 7.9 g of ammonium bicarbonate was added to 100 ml of milli-Q water and mixed, and after refrigeration, refrigerated.

  50 mM ammonium bicarbonate: 1520 μl of milli-Q water was mixed with 80 μl of 1 M ammonium bicarbonate, mixed with light, and then refrigerated.

  Trypsin solution: 20 μl of modified trypsin 100 μg / ml (Promega) and 180 μl of the attached buffer (50 mM AcOH) were mixed and dispensed in small portions and stored at −80 ° C. 20 μl of this trypsin solution (100 μg / ml) and 180 μl of 50 mM ammonium bicarbonate were mixed immediately before use, protected from light and refrigerated.

  Decoloring solution: Immediately before use, 500 μl of 30 mM potassium ferricyanide and 500 μl of 100 mM sodium thiosulfate were mixed and refrigerated.

  Reduction solution: 950 μl of Milli-Q water was mixed with 10 μl of 1 M DDT (Wako Pure Chemical Industries) and 25 μl of 1 M ammonium bicarbonate immediately before use and refrigerated.

  Wash buffer: 3 ml of milli-Q water was mixed with 75 μl of 1 M ammonium bicarbonate immediately before use.

  Alkylating solution: 10 mg of Indocaetamide (Wako Pure Chemical Industries) and 1 ml of washing buffer were mixed immediately before use, and refrigerated after shading.

  Dehydration solution: Immediately before use, 2 ml of 100% acetonitrile (Wako Pure Chemical Industries) and 100 μl of 1 M ammonium bicarbonate were mixed and refrigerated.

  Extract: 1 ml of 100% TFA (Wako Pure Chemical Industries) and 900 μl of MilliQ water were added to a 15 ml Corning tube protected from light, and mixed to obtain a 10% TFA solution. Subsequently, 500 μl of acetonitrile and 500 μl of 10% TFA solution were mixed immediately before use and refrigerated.

  10% formic acid: To a 50 ml Corning tube protected from light, 18 ml of MilliQ water was added, and 2 ml of formic acid (Wako Pure Chemical Industries) was added and mixed in a fume hood.

  To a 15 ml Corning tube protected from 0.1% formic acid, 99.9 ml of milli-Q water and 100 μl of 10% formic acid were added, mixed, and refrigerated.

The used hydrocarbons are as follows.

The used primers are as follows.

Strains used and culture conditions
Rhodococcus bacteria were cultured in IB medium. Static culture is performed at 30 ° C for the R. erythropolis PR4 strain and at 37 ° C for the R. rhodochrous S-1 strain, the R. rhodochrous S-2 strain, the R. rhodochrous R-1 strain, and the R. rhodochrous R-2 strain. All the shaking cultures in the liquid medium were performed at 110 rpm and 28 ° C. In the bilayer culture system, C19 is used as a representative condition for cells to migrate into the alkane, and C12 is used as a representative condition for the cells to adsorb to the surface of the alkane. These alkanes are 5-20% (v / v ) And added to the IB medium and cultured with shaking at 28 ° C. and 110 rpm for 3 days.

Observation by phase contrast microscope 3 μl each of the organic layer and the aqueous layer of the main culture solution to which hydrocarbons were added was collected. This was dropped on a preparation (IWAKI), and a sample with a cover glass (IWAKI) placed thereon was obtained. A sample was placed on the stage of a phase contrast microscope (OLYMPUS DP50), and a drop of immersion oil was dropped on the slide glass. The magnification of the objective lens was set to 100 times, and the observation was first performed in a bright field, and then the observation was performed with a phase difference.

Proteome Analysis Protein Extraction Bacteria obtained from the two-layer culture system were collected by appropriately combining various centrifugations or suction filtration using a hydrophobic filter. 2 μl of sample buffer was added to 1 mg of the collected bacterial cells, mixed, boiled at 100 ° C. for 18 minutes, and sonication was appropriately combined to extract proteins. Then, it cooled to room temperature and centrifuged for 10 minutes at 12000 rpm at 4 degreeC. The obtained supernatant was transferred to another Eppendorf tube, and this solution was used as a protein extract for the next operation.

Measurement of protein concentration The protein concentration was measured using Compat Able ^™ Protein Assay Reagent Set basically according to the instructions. Removal of SDS and mercaptoethanol from the sample was performed as follows. First, the volume of the sample solution was weighed, 5 times the amount of reagent 1 was added, mixed, and allowed to stand for 5 minutes. Next, reagent 2 was added in the same amount as reagent 1, mixed, and centrifuged at 10000 rpm for 15 minutes at 4 ° C. After centrifugation, only the supernatant was removed so as not to collect the resulting precipitate. These operations were repeated once more.

  In order to remove the acid from the precipitate and precipitate the protein, an acetone precipitation method was performed. To the resulting precipitate, 100 μl of milli-Q water and 400 μl of acetone were added and mixed. Then, it was left overnight at −20 ° C. and centrifuged at 12000 rpm for 15 minutes, and the supernatant was removed. The obtained precipitate was applied to a centrifugal evaporator and precipitated and dried.

  Thereafter, 100 μl of denaturing buffer was added and applied to an ultrasonic crusher. The conditions were 30 seconds crushing, 30 seconds interval, and repeated until the precipitate became fine (about 8 times) to dissolve the precipitate. Each sample was centrifuged at 12000 rpm for 15 minutes at 4 ° C, and only the supernatant was collected in a new tube and stored at -20 ° C.

The preparation of the standard was adjusted as follows. 50 μl of the 2 mg / ml BSA standard stock solution supplied with the BCA ^™ Protein Assay Kit was collected in a 1.5 ml tube, and 150 μl of denaturing buffer was added to prepare a 500 μg / ml BSA standard. This 500 μg / ml BSA standard solution is diluted 2 times and prepared to 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.25 μg / ml, 15.625 μg / ml. It was set as the solution.

  Absorbance was measured in the following volume. First, 25 μl of the above standard solution was applied in triplicate to a 96-well plate. Next, the sample was diluted 10, 20, 50, 100, and 200 times in advance with a denaturing buffer, and 25 μl of the sample was similarly applied to a 96-well plate.

  Subsequently, 200 μl of WR was quickly added to each well with a pipetting pipetter, shielded from light with aluminum foil, and incubated at 37 ° C. for 30 minutes. Thereafter, the absorbance at 540 nm was measured with a microplate reader. The protein concentration was calculated from the absorbance value.

SDS-PAGE
First, the electrophoresis kit was washed with 5% EXTRAN MA02 (Merck), 15% T Resolving Gel was set, running buffer was added to hide the gel, and the T Resolving Gel comb was removed. Next, 5 μl of 1 × sample buffer was applied to both ends of the gel, and 5 μl of molecular weight marker and 10 μl of protein extract of each sample were applied to each lane. Electrophoresis was performed at 60 V, and the electrophoresis was terminated when the band reached approximately 5 mm from the bottom of the gel.

  After electrophoresis, the gel was taken out, a micropartel was screwed between two glass plates, the glass was peeled off, and the gel was transferred to a tapper. The staining operation was performed as follows.

  An appropriate amount of fixative was added and shaken for 40 minutes. Subsequently, the fixing solution was discarded, the same amount of washing solution was added, and the mixture was shaken for 10 minutes. Similarly, the washing solution was discarded, an appropriate amount of milli-Q water was added and shaken for 10 minutes, and then the solution was discarded. An appropriate amount of a sensitizing solution was added to this, and after shaking for 1 minute, the solution was discarded. An appropriate amount of milli-Q water was added to the gel after the sensitizing solution treatment, and the mixture was shaken for 1 minute, and the solution was discarded.

  This operation was repeated three times. Subsequently, an appropriate amount of silver nitrate solution was added and shaken at 4 ° C. for 20 minutes, and then the solution was discarded. An appropriate amount of milli-Q water was added thereto, and after shaking for 1 minute, the liquid was discarded. This operation was repeated three times. Finally, an appropriate amount of developer was added, and while carefully observing, the mixture was shaken until the darkness of the band became good, the developer was quickly discarded, and a stop solution was added. This was shaken for 10 minutes, further washed three times with an appropriate amount of milli-Q water, and then the gel was stored in milli-Q water. After dyeing, the gel was sandwiched between OHP films and images were taken with a scanner.

Cutting out and in-gel digestion The gel was cut out as follows. Stained gels stored at 4 ° C. were placed on OHP film matched with graph paper and placed on a light board. The gel was cut into 11 fractions with a scalpel at intervals of 6.5 mm per lane, and the cut gel was cut into 1 mm squares, and the gel pieces were placed in a 1.5 ml Eppendorf tube.

  The decoloring operation was performed as follows. 100 μl of decolorizing solution was added to each sample and shaken at 24 ° C. for 10 minutes using a thermomixer. Thereafter, the decolorization was removed with a pipetman so as not to suck the gel. 500 μl of Milli-Q water was added to these and shaken for 15 minutes at 24 ° C. using a thermomixer to remove Milli-Q water. These operations were repeated three times to completely remove the decolorizing solution.

  In-gel digestion of protein was performed as follows. First, 100 μl of acetonitrile was added using a Pasteur pipette, and after shaking for 5 minutes using a thermomixer, acetonitrile was similarly removed using a Pasteur pipette.

  A tube lid that had been previously pierced with a syringe in the tube lid was prepared and replaced with a sample tube lid. These were centrifuged for 15 minutes with a centrifugal evaporator, and the liquid was evaporated to dry the gel.

  Here, the lid with the hole was taken out and returned to the original lid. 100 μl of the reducing solution was added to these, and the mixture was shaken at 56 ° C. for 60 minutes. 100 μl of washing buffer was added to these samples and shaken at 24 ° C. for 10 minutes, and the liquid was similarly removed.

  Subsequently, 100 μl of the alkylating solution was added and shaken at 24 ° C. for 45 minutes while shielding light, and then the solution was removed. To this, 100 μl of washing buffer was added and shaken at 24 ° C. for 10 minutes. Then, 200 μl of dehydrated solution was added and shaken at 24 ° C. for 10 minutes, and then all the solutions were removed. Furthermore, 200 μl of dehydrating solution was added to these samples, shaken at 24 ° C. for 10 minutes, and the solution was discarded. Subsequently, the sample was dried as described above.

  To the dried sample, 25 μl of trypsin solution was added and left on ice for 30 minutes to soak the gel piece with the trypsin solution. If necessary, excess trypsin solution was removed. These samples were reacted overnight (about 15 hours) at 37 ° C.

  After completion of the reaction, 50 μl of the extract was added and shaken at 24 ° C. for 30 minutes. After flushing, the solution was carefully removed so as not to contain gel pieces and transferred to a new tube. This extraction operation was repeated once more. The recovered protein extraction solution was stored at −20 ° C.

Formic acid treatment
Each sample stored at -20 ° C was removed and the lid was replaced with a lid that had been pre-drilled. The solution was centrifuged with a centrifugal evaporator until the solution was completely evaporated (about 1 hour 30 minutes). Here, the lid with the hole was taken out and returned to the original lid.

13 μl of 0.1% formic acid was added to these samples and mixed by vortexing. Thereafter, it was flushed with a centrifuge and stored at -20 ° C. (The operation so far was performed the day before LC / MS / MS.)
On the day of subjecting to LC / MS / MS, the sample was vortexed, flushed, and stored on ice until the sample was applied.

nanoLC-ESI-MS / MS measurement
The Microbore HPLC system used was Paradigm MS4 (Michrom Bioresources). The column was Magic C18 (200 A, 3 μm, 0.2 × 50 mm; Michrom Bioresources) with a spray needle (AMR). As the solvent, buffer A (2% vol / vol acetonitrile, 0.1% formic acid) and buffer B (90% vol / vol acetonitrile, 0.1% formic acid) were used. Peptide separation / elution was performed with a linear gradient of 5-65% buffer B in 20 minutes.

  The peptide extract was first adsorbed and desalted on a C18 cartridge (Michrom Bioresources) and then led to the analytical column using a switching valve. The column eluate was directly ionized by an electrospray ionization source (AMR), and subjected to mass spectrometry in a positive mode using an LCQ Deca XP ion trap mass spectrometer (Thermo Fisher). Peak detection was performed in the range of m / z 500-2000 using Xcalibur software (Thermo Fisher).

Analysis of spectral data
MS / MS data was analyzed using SEQUEST 1, 2 (Thermo Fisher). Peptide identification uses sequence information with a correlation factor (Xcorr) value of 2.0 or higher for bivalent peptides and 2.5 or higher for trivalent peptides, and sequence information with a final score (Sf) value of 0.85 or higher. It was. Spectral data were searched against PR4 genomic database.

Extraction of total DNA Strains were planted on IB agar medium with platinum ears and cultured at 30 ° C. for 4-5 days (2 per sample). The cells were collected, transferred to a falcon tube, and suspended in 5 ml of TE buffer per 1 g of wet cell weight. Lysozyme was added to the cell suspension to a final concentration of 4 mg / ml, and the cells were cultured at 30 ° C. until the bacterial body became viscous (1 hour or longer). To this sample solution, 0.5 M EDTA solution (final concentration 0.1 M) and proteinase K (final concentration 50 μg / ml) were added, followed by incubation at 30 ° C. for 10 minutes.

  Thereafter, a 20% SDS solution was added to this sample solution to a final concentration of 1%, and the mixture was immediately stirred by inversion and cultured at 37 ° C. Next, add a neutral phenol solution (pH 8.0) saturated with an equal volume of Tris-HCl, gently invert and stir for 5 minutes, then centrifuge at 12000 rpm for 10 minutes at 4 ° C to cut the tip. Was slowly sucked up and transferred to a new container.

  Next, to this sample solution was added an equivalent amount of a neutral phenol and chloroform solution (chloroform and isoamyl alcohol mixed at a volume ratio of 24: 1) to the sample in an equal amount, and the mixture was gently inverted by stirring for 5 minutes, then at 12000 rpm. Centrifugation at 4 ° C. for 10 minutes, the upper layer was slowly sucked up using a tip with a tip, and transferred to a new container. This operation was repeated until the white intermediate layer disappeared.

  After the intermediate layer has disappeared, add an equal volume of chloroform solution to the sample, gently invert and stir for 5 minutes, then centrifuge at 12000 rpm for 10 minutes at 4 ° C. Slowly aspirate the upper layer using a tip with a tip cut Moved to a new container.

  To this sample solution, 1/10 volume of 3M sodium acetate solution and an equivalent amount of isopropanol solution were added, mixed slowly, and then incubated at -80 ° C for 30 minutes. This sample solution was centrifuged at 12000 rpm for 10 minutes at 4 ° C., and the supernatant was removed to collect a white precipitate.

  An appropriate amount (about 5 ml) of 70% cold ethanol was added to this precipitate and washed carefully, followed by centrifugation at 12000 rpm for 10 minutes at 4 ° C., and the supernatant was removed to recover a white precipitate. The container containing the precipitate was air-dried by turning it upside down, dissolved in an appropriate amount of TE buffer (1 ml), and transferred to a 2 ml Eppendorf tube.

  To this sample solution, RNase A was added to a final concentration of 40 μg / ml and cultured at 37 ° C. for 1 hour. The sample solution after RNase treatment was treated with phenol and chloroform as described above, isopropylated, rinsed with 70% ethanol, and dissolved in TE buffer. Thereafter, a part of the sample solution was taken and subjected to electrophoresis at 100 V using a 0.8% agarose gel to confirm that the target DNA was extracted.

Extraction of plasmid DNA
Extraction of plasmid DNA from Rhodococcus One portion of the fungal toothpick on an agar plate medium was suspended in sterilized milli-Q water and centrifuged at 15000 rpm for 10 minutes at 4 ° C. to recover the precipitate. To the precipitate, 100 μl of Solution I having a final lysozyme concentration of 10 mg / ml was added and incubated at 37 ° C. for 30 minutes, and then 50 μl of Solution II was further added and cultured at 75 ° C. After cooling to room temperature, 15 μl of acidic phenol was added and stirred. The supernatant was recovered by centrifugation at 15000 rpm for 10 minutes at 4 ° C.

  To this sample solution was added an equivalent amount of neutral phenol and chloroform solution (chloroform and isoamyl alcohol mixed at a volume ratio of 24: 1) and the sample in an equal amount, and after tumbling for 5 minutes, 4 minutes at 12000 rpm for 4 minutes. Centrifugation was performed at 0 ° C., and the upper layer was slowly sucked up using a tip with a tip cut and transferred to a new container. This operation was repeated until the white intermediate layer disappeared.

  After the intermediate layer has disappeared, add an equal volume of chloroform solution to the sample, invert and stir for 5 minutes, then centrifuge at 12000 rpm for 10 minutes at 4 ° C. Slowly aspirate the upper layer using a tip with a tip, Moved to a new container.

  To this sample solution, 1/10 volume of 3M sodium acetate solution and an equivalent amount of isopropanol solution were added, mixed slowly, and then incubated at -80 ° C for 30 minutes. This sample solution was centrifuged at 12000 rpm for 10 minutes at 4 ° C., and the supernatant was removed to collect a white precipitate.

  An appropriate amount (about 5 ml) of 70% cold ethanol was added to this precipitate and washed carefully, followed by centrifugation at 12000 rpm for 10 minutes at 4 ° C., and the supernatant was removed to recover a white precipitate. The container containing the precipitate was air-dried by turning it upside down, dissolved in an appropriate amount of TE buffer (1 ml), and transferred to a 2 ml Eppendorf tube.

To this sample solution, RNase A was added to a final concentration of 40 μg / ml and cultured at 37 ° C. for 1 hour. The sample solution after RNase treatment was treated with phenol and chloroform as described above, isopropylated, rinsed with 70% ethanol, and dissolved in TE buffer. Thereafter, a part of the sample solution was taken and electrophoresed at 100 V using a 0.8% agarose gel to confirm the plasmid DNA.

Extraction of plasmid DNA from E. coli Thermal alkali method A portion of the toothpick on the agar plate was suspended in sterilized milli-Q water, and centrifuged at 15000 rpm for 10 minutes at 4 ° C to collect the precipitate. To the precipitate, 100 μl of Solution I having a final lysozyme concentration of 10 mg / ml was added and incubated at 37 ° C. for 30 minutes, and then 50 μl of Solution II was further added and cultured at 75 ° C. After cooling to room temperature, 15 μl of acidic phenol was added and stirred. Centrifugation was performed at 15,000 rpm for 10 minutes at 4 ° C., and the supernatant was recovered and electrophoresed at 100 V using a 0.8% agarose gel to confirm plasmid DNA.

Plasmid extraction using Wizard (R) SV Minipreps DNA Purification System
Wizard® SV Minipreps DNA Purification System (Promega) was used. The operation was basically performed according to the instructions. The bacterial cells were scraped from the plate with a platinum loop, suspended in milli-Q water, and centrifuged at 15000 rpm for 10 minutes at 4 ° C. to recover the precipitate. Next, 250 μl of Cell Resuspension Solution was added and suspended, 250 μl of Cell Lysis Solution was added, and the mixture was stirred by inversion four times. Next, 10 μl of Alkaline Protease Solution was added, and the mixture was tumbled 4 times and allowed to stand for 5 minutes.

  Next, Neutralization Solution was added, and the mixture was stirred by overturning 4 times and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The supernatant was transferred to a tube with a spin column and centrifuged at 15,000 rpm, 4 ° C. for 1 minute. The solution in the tube was discarded, 750 μl of Wash Solution was added to the spin column, and centrifuged at 15,000 rpm, 4 ° C. for 1 minute.

  The solution in the tube was discarded, 250 μl of Wash Solution was added to the spin column, and centrifuged at 15,000 rpm at 4 ° C. for 1 minute. The solution in the tube was discarded and centrifuged at 15,000 rpm, 4 ° C. for 2 minutes. The spin column was transferred to a new tube, 100 μl of Nuclease-Free Water was added, and the mixture was centrifuged at 15,000 rpm, 4 ° C. for 1 minute. The solution was recovered and electrophoresed at 100 V using a 0.8% agarose gel to confirm plasmid DNA.

DNA recovery from agarose gels
QIAquick® GelExtraction Kit (QIAGEN) was used. The operation was basically performed according to the instructions. Electrophoresis of the DNA solution was performed using an agarose gel. The part of the target mobility was cut out using a cutter knife on a transilluminator. The gel cut out in a 2 ml Eppendorf tube was added, and 3 times the volume of Buffer QG was added to the gel and reacted at 50 ° C. for 10 minutes. After the gel slice was completely dissolved, the same volume of isopropanol as the gel was added and stirred.

  The sample solution was added to a tube with a spin column and centrifuged at 15,000 rpm, 4 ° C. for 1 minute. The solution was discarded, 500 μl of Buffer QG was added, and the mixture was centrifuged at 15,000 rpm, 4 ° C. for 1 minute. The solution was discarded, 750 μl of Buffer PE was added, and the mixture was centrifuged at 15,000 rpm, 4 ° C. for 1 minute. The solution was discarded and centrifuged at 15,000 rpm, 4 ° C. for 1 minute.

  The spin column was transferred to a new tube, 50 μl Buffer EB was added to the center of the membrane, and centrifuged at 15,000 rpm at 4 ° C. for 1 minute. In order to confirm that the target DNA fragment was cut out, electrophoresis was performed using a 0.8% agarose gel.

DNA amplification by PCR
1 μl of total DNA, 4 μl of dNTP, 5 μl of buffer, 0.5 μl of EX taq, 1 μl each of Forward Primer and Reverse Primer, and 37.5 μl of sterile milliQ water in a PCR tube, total 50 μl reaction A system was created. PCR reaction was performed at 95 ° C. for 10 min, (95 ° C. for 1 min → 70 ° C. for 1 min → 72 ° C. for 2 min) × 30, 72 ° C. for 10 min. The annealing temperature was appropriately changed from 60 ° C to 75 ° C. After PCR, 5 μl of the sample was taken and subjected to confirmation electrophoresis using 0.8% or 1.6% agarose gel.

DNA sequence Sequence reaction
The DNA solution was heat denatured at 100 ° C. for 10 minutes and rapidly cooled on ice water. 2 μl to 11.8 μl of denatured DNA in a tube for PCR, 3 μl of buffer, 2 μl of Big Dye, and sterilized milli-Q water were appropriately added to prepare a total reaction system of 20 μl. The sequence reaction was preheated to 96 ° C. in advance (96 ° C. 30 sec → 50 ° C. 15 sec → 60 ° C. 4 min) × 24.

Purification of the sequencing reaction Transfer the sample solution after the sequencing reaction to a 1.5 ml Eppendorf tube, add 5 μl of 125 mM EDTA, 60 μl of 99.5% ethanol, stir by inversion, let stand at room temperature for 15 minutes, 15,000 rpm, 4 ° C And centrifuged for 20 minutes. The supernatant was removed, 70% ethanol was added, and the mixture was inverted and stirred, and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The supernatant was removed and the sample was air dried and stored at -20 ° C. protected from light.

Purification of sequence reaction Sequence analysis was performed with ABI Prism model 3100 automatic sequencer (Applied Biosystems). This step was commissioned to the Research Institute for Biological Resources Science at Nihon University. Homology search for DNA sequences is available on the Internet at the National Center for Biotechnology Information (NCBI) [HP address:
1252304127890_5
] BLAST Program [Standard nucleotide-nucleotide BLAST (
1252304127890_6
)].

Cloning
TA Cloning Using TOPO TA Cloning® Kits TOPO TA Cloning® Kits (Invitrogen) were used for cloning. The operation was basically performed according to the instructions. A DNA solution (1 μl to 4 μl), a salt solution (1 μl), a TOPO (R) Vector (1 μl), and sterilized milli-Q water were added to prepare a total 6 μl reaction system. The reaction was allowed to proceed for 30 minutes at room temperature, and then the reaction system was placed on ice, and the entire amount was added to the one shot (R) Chemically Competent E. coli previously pipetted into a culture tube. Place on ice for 30 minutes and then warm to 42 ° C.

  250 μl of SOC medium was added at room temperature, and cultured with shaking at 37 ° C. for 1 hour. Centrifuge at 15000 rpm for 10 minutes at 4 ° C, discard the liquid until the precipitate disappeared, suspend and suspend the whole volume in advance (Amp 100 μg / ml, LB medium with X-gal, IPTG) The cells were smeared and inoculated overnight at 37 ° C. After culturing, the colonies were isolated and stored using a sterilized toothpick.

Ligation
TakaRa Ligation kit ver.2.1 (Takara Shuzo Co., Ltd.) was used. The operation was basically performed according to the instructions. Equal amounts of DNA solution and I solution were added to a 1.5 ml Eppendorf tube, and mixed gently with a pipette for a total of 20 μl and reacted at 16 ° C. for 30 minutes. The reaction solution was used as it is or after purification by ethanol precipitation as necessary.
transformation
Transformation of E. coli Competent cells were JM109 COMPETENT high (TOYOBO) or HB101 COMPETENT high (TOYOBO). The operation was basically performed according to the instructions. Competent cells were dissolved in ice water, DNA to be transformed was added, and the mixture was allowed to stand in ice water for 30 minutes. Warmed at 42 ° C. for 30 seconds and cooled in ice water for 2 minutes. Next, 900 μl of SOC medium was added, and cultured with shaking at 37 ° C. for 1 hour.
Centrifuge at 15000 rpm for 10 minutes at 4 ° C, discard the liquid until the precipitate disappears, suspend and suspend the whole volume in advance (Amp 50 μg / ml or Km 50 μg / ml, X-gal LB medium containing IPTG), and the cells were inoculated and cultured overnight at 37 ° C. After culturing, the colonies were isolated and stored using a sterilized toothpick.

Electroporation The cells were inoculated with 5 ml of IB liquid medium with a platinum loop and cultured at 110 rpm and 28 ° C. for 3 to 5 days (preculture). 5-15 μl (or turbidity of 0.01-0.05) of the preculture was replanted with 5 ml of fresh IB liquid medium. At this time, penicillin G potassium was added to the culture solution to a final concentration of 0.1 μg / ml depending on the strain. The culture was continued at 110 rpm and 28 ° C. for about 10 to 20 hours until OD ₆₆₀ = 0.42 to 0.8 (main culture).

  The culture solution was transferred to a 15 ml centrifuge tube and centrifuged at 12,000 rpm at 4 ° C. for 10 minutes to collect the cells. 5 ml of ice-cooled sterilized milli-Q water was added to the precipitate, suspended, and centrifuged at 12,000 rpm at 4 ° C. for 10 minutes to collect the precipitate.

  5 ml of ice-cooled 10 mM sucrose solution was added to the precipitate, suspended, and centrifuged at 12,000 rpm at 4 ° C. for 10 minutes to collect the precipitate. This operation was performed twice. Next, 1 ml of ice-cold sucrose solution was added to the precipitate, suspended, transferred to a 1.5 ml microtube, and centrifuged at 15,000 rpm at 4 ° C. for 10 minutes to collect the precipitate.

  To the precipitate, 40 ml of ice-cold sucrose solution was added and suspended, 5 to 20 μl of a DNA sample was added, and the mixture was kept on ice for 15 minutes. After transferring the cell-DNA mixture to an ice-cooled cuvette and setting it in the chamber, electrolysis was performed under the conditions of an applied voltage of 1.50 kV, a resistance of 400 Ω, and a capacitance of 25 μFD. Polled.

  The whole cuvette was kept on ice water for 10 minutes, and 360 μl of IB liquid medium was added into the cuvette. Transfer to a sterile plastic test tube using a sterile Pasteur pipette and add 600 μl of IB liquid medium. After culturing at 110 rpm and 28 ° C. for 2 to 4 hours, concentrating by centrifugation, the cells were plated on IB agar medium (containing various antibiotics) and cultured at 37 ° C. for 2 to 4 days.

Comparison of growth of C12 by addition culture using liquid medium Each strain was inoculated into IB liquid medium by one platinum loop and cultured at 28 ° C. with shaking for 3 days. This preculture was assumed to be 10 ⁹ cfu / ml, diluted appropriately to 10 ⁴ cfu / ml, and taken into a new medium contained in a φ24 test tube (IWAKI GLASS). Subsequently, C12 was added to a final concentration of 5% (v / v), and cultured with shaking at 28 ° C. and 110 rpm.

These cultures were sampled every 24, 48, 72, 96, and 144 hours, and the viable cell count was measured. The experiment was performed as follows. The test tube was suspended by vortexing, and 100 μl of the suspension was quickly taken, and added to a 1.5 ml eppen containing 900 μl of IB liquid medium to make a 10 ⁻¹ dilution.

This was made up to a ^10-7 dilution. Three dilutions of the 10 ⁻⁵ to 10 ⁻⁷ dilutions were each spread on 100 μl of IB agar medium and spread. The cells were cultured at 30 ° C. for 2 days (until colonies were confirmed), and the number of bacteria was measured.

Examination of related proteins by proteome analysis Search for specific expressed protein C19 as a representative condition for cells to transfer to the inside of alkane in a control and bilayer culture system without alkane, C12 as a representative condition for cells to adsorb on the surface of alkane As a result of SDS-PAGE under three conditions using, no significant difference was observed in the band pattern (Fig. 3).

  Subsequently, each lane was cut out at an interval of 6 mm, subjected to in-gel digestion with trypsin, and then subjected to LC-MS / MS analysis. Table 6 summarizes the proteins detected by LC-MS / MS analysis at 1% or more under C19 addition conditions. In the obtained data, we focused on 1) GroEL2 which occupies 1% or more of the total detected protein, 2) the amount of protein increased by C19 addition compared to control, and 3) which was detected with good reproducibility.

  The% column value in the table indicates the detected ratio of each protein, and the C19 / N column value indicates the rate of increase compared to the control without addition of alkane.

The protein groups detected by LC-MS / MS are as follows.

  GroEL is a type of molecular chaperone that is known to help protein folding, and its expression is induced under various conditions such as heat stress, nutrient deficiency, and infection (Jenkins 1991; J. Bacteriol, 173: 1992). -1996, Young 1989; Cell, 59: 5-8). In E. coli, GroEL is known to be essential for growth at all temperatures (Fayet 1989; J. Bacteriol, 171: 1379-1385).

  GroEL has a 14-mer structure in which a ring consisting of seven subunits with a molecular weight of 57 kDa overlapped back to back, forming a complex with the co-chaperone GroES (Xu 1997; Nature, 388: 741- 750) and 10 to 15% of the total protein is known to fold (Ewalt 1997; Cell, 90: 491-500).

  Other Gram-positive bacteria with high GC content, such as Corynebacterium bacteria and Streptomyces bacteria, have two GroELs, and GroEL2 is known to exist apart from the GroESEL operon and not to form operons with other genes. (Barreiro 2005 J. Bacteriol, 187: 884-889, Servant 1993 Gene, 134: 25-32).

  The functions of GroEL1 and GroEL2 were unknown, but a paper published in Cell in 2005 succeeded in destroying GroEL1 in Mycobacterium, and the disrupted strain inhibited biofilm formation. (Anil 2005). Thus, we focused on GroEL2 detected by LC-MS / MS.

Cloning of groEL2 gene of PR4 strain
Using the DNA of PR4 strain, a sequence containing the groEL2 gene was amplified with a combination of primer 366F-11 and GroEL-R1 to obtain a DNA fragment having a target molecular weight (FIG. 4). This PCR product was TA cloned using TOPO TA Cloning® Kits (FIG. 5, lane 4). The sample obtained by treating the cloned plasmid with EcoRI for the insert check (FIG. 5, lane 5) was compared with the PCR product of the groEL2 gene used for the insert (FIG. 5, lane 1).

  As a result, both had a band of the same size around 1.9 kb, so it was determined that the groEL2 gene was cloned into the topovector.

  Next, the cloned plasmid was cloned into the EcoRI site of pK4, which is a Rhodococcus-E. Coli shuttle vector, using a sample treated with EcoRI.

  Compare the cloned plasmid treated with EcoRI (Figure 6, lane 3) to the insert check and the plasmid with the groEL2 gene cloned in the topovector treated with EcoRI (Figure 6, lane 1) did.

  As a result, since both had a band of the same size in the vicinity of 1.9 kb, it was determined that the groEL2 gene was cloned into pK4. In addition, the obtained plasmid was treated with KpnI and ApaI (Fig. 6 (A), lane 4), and the orientation was confirmed. Transcription of the Km resistance gene of pK4 so that the insert was transcribed by Km pressure. The plasmid [Fig. 6 (B) (I)] cloned in the same orientation was used for the next operation.

Introduction of PR4 of groEL2 gene
A plasmid (pK4 + groEL2) containing PR4 strain groEL2 gene was introduced into PR4 strain by electroporation. The obtained transformant was cultured in a two-layer culture system, and the localization of the cells was observed. The results under each condition are shown in FIG. 7, and the interaction with a series of alkanes is shown in FIG. 8, FIG. 17, and FIG.

  As a result, under conditions where alkanes of C15 or higher were added, transfer into the alkane particles was observed as in the parent strain (FIG. 9), but under conditions where alkanes of C10-C14 were added, Cells adsorbed on the surface and cells that migrated into the particles were observed simultaneously (FIG. 10).

  In addition, under the conditions where C7 and C8 were added, cells could not be observed in the parent strain, whereas the transformant was observed to be adsorbed on the surface of the alkane particles (FIG. 11). From this, it was speculated that the introduction of groEL2 gene changed the interaction with alkane, and it was able to grow even under the condition where C7 and C8 were added.

  Since it was considered that the introduction of the groEL2 gene improved locality and growth as described above, a growth curve under the condition of adding C12 was made and confirmed as a representative.

  As a result, the parent strain and the transformed strain showed the same growth at the initial stage of culture, but the number of viable bacteria in the transformed strain started to increase after 50 hours, and finally the number of viable bacteria increased about 100 times. Okay (Figure 12). These results suggest that GroEL2 is involved in the determination of cell localization and growth.

Introduction of groEL2 gene derived from PR4 into other strains
A plasmid containing groEL2 gene of PR4 strain was introduced into the same genera, R. rhodochrous S-1, S-2, R-1 and R-2. The obtained transformant was cultured in a two-layer culture system with the addition of C12 and C19, and each parent strain was compared with the transformant in order to confirm whether the cell localization had changed (FIG. 13). FIG. 14, FIG. 15, FIG. 16).

  As a result, in the R-1 strain, cells could not be observed in the parent strain under the condition where C12 was added, whereas the transformed strain was observed to be adsorbed on the surface of the alkane particles [FIG. ), (II)].

  As with the PR4 strain, it was speculated that the introduction of the groEL2 gene changed the interaction with alkane, and it was able to grow even under the condition where C12 was added. On the other hand, under the condition where C19 was added, it was observed that the parental strain and the transformed strain had both surface adsorption and internal transfer to alkane, and there was no change [Fig. 13 (III), (IV )].

  In the R-2 strain, the parental strain and transformation hardly grew under the condition of C12 addition [Fig. 14 (I), (II)]. Also, under the condition of C19 addition, alkane was adsorbed on the surface and transferred to the inside. There was no change observed in the mixture [Fig. 14 (III), (IV)].

  In the S-1 strain, the parent strain and the transformation were observed to have a mixture of surface adsorption and internal transfer to alkanes under the conditions of C12 addition and C19 addition (FIG. 15). ).

  In the S-2 strain, it was observed that the parent strain and transformation were adsorbed on the surface of alkane under the conditions of C12 addition and C19 addition, and there was no change (FIG. 16).

  In the R-1 strain, the transformed strain was able to grow under the condition of adding C12 that was not able to grow in the parent strain. Thus, it was confirmed that GroEL2 of PR4 strain was also involved in the growth of other species.

Examination of the influence of magnesium chloride The influence of the Mg salt in the IB medium on the localization and growth of the transformant was examined. Rhodococcus erythropolis introduced with GroEL2 gene was used as a trait development.

  When comparing the characteristics of the transformant using the IB medium supplemented with magnesium chloride described above and the characteristics of the transformant using the IB medium substantially free of magnesium chloride, as shown in FIG. In an environment where C6 hydrocarbons were added to the IB medium, the former transformant could not grow, whereas the latter transformant could grow.

  That is, by reducing the concentration of the inorganic salt contained in the medium of the transformant as much as possible, the transformant can grow even in an environment where lower carbon number hydrocarbons are present, and it can be decomposed and metabolized. it can.

  If Mg chloride is added at 44.3 micromolar under the condition where C6 is added, PR4 (pK4 + EL2) cannot grow, so this concentration seems to be the upper limit.

According to this embodiment, by further reducing the MgCl ₂ concentration, the localization of the bacteria tends to change from the transfer type to the adsorption type, and when the concentration is around 8.9 mM, The ratio of the adsorption type was reversed, and at a concentration lower than that, the ratio of the transfer type cells increased.

In addition, in order to examine whether the phenomenon described above is a phenomenon peculiar to magnesium chloride, the above-mentioned medium containing only K ₂ HPO ₄ and (NH ₄ ) ₂ SO ₄ is added with magnesium sulfate, magnesium nitrate, magnesium acetate, lactic acid. Magnesium was added to the same concentration as magnesium chloride in the IB medium, and localization was examined in the presence of C12. As a result, even when other magnesium salts such as magnesium sulfate and magnesium nitrate were added, the cells were adsorbed on the surface of the C12 particles like magnesium chloride.

Thus, in order to metabolize lipophilic solvents using Rhodococcus bacteria, among the inorganic salts added to the bacterial medium, the localization of the bacteria with respect to the lipophilic solvent is also affected. By adjusting the concentration, it was possible to change from a form that adsorbs bacteria to the solvent to a form that transfers to the solvent.

The concentration of these inorganic salts such as magnesium is 88.5 nM or less, preferably 8.9 nM or less, more preferably 0.9 nM or less, from the viewpoint of controlling the localization of bacteria.

Claims (7)

groEL2 genes is reacted with the introduced bacteria carbon atoms belonging to the genus Rhodococcus 14 following hydrocarbons by transformation, decompose the hydrocarbon, metabolize, how to process the hydrocarbons. The method for treating hydrocarbons according to claim 1, wherein the bacterium is Rhodococcus erythropolis PR4. The method for treating hydrocarbons according to claim 1, wherein the hydrocarbon has 7 to 14 carbon atoms. The method for treating hydrocarbons according to claim 1, wherein the bacteria are cultured in a medium having a limited inorganic salt concentration to decompose and metabolize the hydrocarbons . The hydrocarbon treatment method according to claim 4 , wherein the inorganic salt is a magnesium salt. The hydrocarbon treatment method according to claim 5 , wherein the concentration of the magnesium salt is limited to 44.3 μM or less. The hydrocarbon treatment method according to claim 6 , wherein the concentration of the magnesium salt is limited to 88.5 nM or less.