KR100465853B1 - Method for improving viability by cold adaptation of Bradyrhizobium japonicum - Google Patents

Abstract

The present invention relates to a method of enhancing prebiotics by low temperature pretreatment of Bradyrhizobium japonicum . The present invention relates to a method for enhancing viability by increasing resistance to various environmental stresses. Low temperature pretreatment of the bradyrizobium japonicum of the present invention to enhance the viability of the strain increases the resistance to the stress received during the formulation of the strain can be usefully used in the preparation of microbial fertilizers using the strain have.

Description

Method for improving viability by cold treatment of Bradyrizobium japonicum strain {Method for improving viability by cold adaptation of Bradyrhizobium japonicum}

The present invention relates to a method of enhancing prebiotics by low temperature pretreatment of Bradyrhizobium japonicum , and more particularly, by treating low temperature of Bradyrizobium japonicum with high temperature, drying, alcohol and oxidative stress. The present invention relates to a method for enhancing viability by increasing resistance to various environmental stresses.

Many studies have been conducted on the effect of low temperature pretreatment on the environmental stress of microorganisms (Crist et al., Appl. Environ. Microbiol , 1984, 47: 895-900, Panoff et al., Crybiol , 1995, 32: 516- 520). When E. coli cultured at 37 ° C. was frozen for 6 hours at 10 ° C. after low temperature pretreatment, it was reported that the activity of microorganisms was higher (Goldstein et al., Proc. Natl. Acad. USA. 1990, 87: 283-287). Also has a much larger viable force in the freezing conditions in the case of low temperature pretreatment by Bacillus interference Tiller's (Bacillus subtilis) and Lactococcus lactis subspecies lactis low temperature pretreatment than no cold pretreatment at (Lactococcus lactis subsp. Lactis) ( Panoff et al., Cryobiol, 1995, 32: 516-520, Willimsky et al., J. Bacteriol. 1990, 87: 5589-5593). Low temperature pretreatment of Enterococcus faecalis has been reported to increase cryotolerance (Thammavongs et al., Lett. Appl. Microbiol . 1996, 23: 398-402). In addition, microorganisms recovered in batch cultures have been shown to have higher activity than microorganisms when pretreated at low temperatures (Hartke et al., Appl. Environ. Microbiol . 1994, 60: 3474-78).

However, such a phenomenon is not common. For example, the resistance to freezing of 259 strains, including 32 genera and 135 species of microorganisms, is specific to some species and some psychrophilic microorganisms are known to be rather sensitive to freezing storage (Wamasato et al. , Cryobiol . 1973, 10: 453-463. Thiobacillus ferrooxidans pretreated at 25 ° C. to 5 ° C. did not show any effect on freezing conditions at −15 ° C. (Hubert et al., Curr. Microbiol . 1994, 28: 179-183 In the lactic acid bacteria, not a few strains showed resistance when cold pretreated at 10 ° C. (Kim and Dunn, Curr. Microbiol . 1997, 35: 59-83; Kim et al., Cryobiol. 1998, 37: 86-91).

When the microorganism is subjected to low temperature pretreatment, a specific reaction (defense mechanism) is caused by cold shock, which is largely divided into biochemical reactions and defense mechanisms at the molecular level. Common biochemical reactions include structural changes in cell membranes, and defense mechanisms at the molecular level include cold shock protein (CSP) (Fujii et al., FEMS Microbiol Lett . 1999, 178: 123-128; Graumann et al., J.) . Bacteriol 1996, 178: 4611-4619; Horton et al., Antonie van Leeuwenhoek , 2000, 77: 13-20) and mechanisms for producing specific proteins such as cold acclimation protein (CAP) are known (Drouin et al. , FEMS Microbiol.Ecol. 2000, 32: 111-120) The specific mechanism of action is still unknown. Thus, it is necessary to understand the mechanism by which low temperature pretreatment reacts to stress, which may consequently help to successfully control the growth and maintenance of microorganisms under environmental stress.

Rhizobia, a nitrogen-fixed strain, is gram-negative and has motility. When legumes are infected by lysovia, they form root nodule and gaseous nitrogen (N ₂ ) is combined with hydrogen. Nitrogen fixiation is performed to convert to nitrogen (NH ₃ ). Nitrogen fixation in the symbiotic relationship between legumes and lysvia is of great agricultural importance, and the nitrogen in the form of compounds in the soil is, in fact, the result. In the case of unfertilized soil, nitrogen deficiency is a natural result, but under these soil conditions, legumes with root nodules solve the problem of nitrogen deficiency themselves, so they can grow under soil conditions where plants cannot grow due to nitrogen deficiency. Stacey et al., Mol. Plant-Microbe Interact . 1991, 4: 332-340. If you try to formulate such a nitrogen-fixed strain is subjected to a lot of stress in the formulation process, the viability is sharply dropped. However, there is no report on how to enhance the viability of the nitrogen-fixed strain to date.

Accordingly, the present inventors confirmed that the low temperature treatment of Bradyrizium japonicum has an increased resistance to environmental stress (high temperature, dryness, alcohol, and oxidative), and the microorganism has a viability of the microorganism even after the formulation process. The present invention was completed by confirming maintenance.

It is an object of the present invention to provide a method for enhancing the viability by pretreating Bradyrizium japonicum at low temperature.

Figure 1 is a schematic diagram of the experiment to determine whether the resistance to environmental stress after cold pretreatment of Bradyrizium japonicum strain,

① Incubate until late growth or early stagnation before strain pretreatment

② pretreatment for 16 hours,

③ Heat (28 ℃, 42 ℃), hydrogen peroxide (100 ppm, 500 ppm, 1,000 ppm), ethanol (5%, 10%) or dry stress,

* Chloramphenicol Treatment

Figure 2 shows the survival rate when the high temperature (42 ℃) stress to the low temperature pretreated Bradyrizium japonicum strain and the control group,

●, ■: low temperature pretreatment, ○, □: control,

●, ○: treatment at 28 ℃, ■, □: treatment at 42 ℃,

Figure 3 shows the survival rate when alcohol stress was given to the low-temperature pretreated Bradyrizobium japonicum strain and the control group,

●, ■: low temperature pretreatment, ○, □: control,

●, ○: 5% ethanol treatment, ■, □: 10% ethanol treatment,

Figure 4 shows the survival rate when given oxidative stress to the cold pretreated Bradyrizium japonicum strain and the control group,

●, ■, ▲: low temperature pretreatment, ○, □, △: control,

●, ○: 100 ppm hydrogen peroxide treatment, ■, □: 50 ppm hydrogen peroxide treatment,

▲, △: 1,000 ppm hydrogen peroxide treatment,

5 shows the survival rate when dry stress was applied to the low-temperature pretreated Bradyrizobium japonicum strain, the control group or the chloramphenicol-treated strain,

●: low temperature pretreatment, ■: control, ▲: chloramphenicol treatment,

6 is an electrophoresis photograph of PCR amplifying the csp gene from genome DNA of Bradyrizobium japonicum strain and Escherichia coli ,

M: size marker, 1: Bradyrizium japonicum, 2: E. coli,

FIG. 7 compares partial amino acid sequences of Bradyrizobium japonicum strains, E. coli and Csp protein derived from Sinorhizobium meliloti .

In order to achieve the above object, the present invention provides a method for enhancing the bio-viability by low-temperature pretreatment of Bradyrizium japonicum.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for enhancing the bioburden by low temperature pretreatment of Bradyrizium japonicum.

It is preferable that it is 4-15 degreeC, and, as for the temperature at the time of low temperature treatment above, it is more preferable that it is 4 degreeC. In addition, the low temperature treatment time is preferably 6 to 24 hours, more preferably 16 hours.

Low temperature pretreatment of Bradyrizium japonicum as described above increases the viability of the strain by having resistance to other environmental stresses such as high temperature, drying and oxidative stress.

As a result of confirming the effect of improving the resistance to high temperature stress of low temperature pretreatment, the low temperature pretreatment when exposed to high temperature maintains more than 100 times the viability compared to the low temperature pretreatment. In addition, when exposed to high temperature for 1 day, low temperature pretreatment showed a reduction rate of 100 times or more than the initial cell count, whereas low temperature pretreatment showed a decrease of 1,000 times or more. It can be seen that the resistance to this increases (see FIG. 2 ). In addition, as a result of confirming the effect of low temperature pretreatment on alcohol stress, the viability increased more than 1,000 times compared with the control group, and the low temperature pretreatment decreased 100 times by 4 hours when exposed to 5% concentration of alcohol, and the control group was 10,000 times. In the case of low temperature pretreatment even at 10% alcohol, it was confirmed that the viable cell count was maintained for 2 hours or more (see FIG. 3 ).

The low-temperature pretreatment of Bradyrizobium japonicum also increased resistance to oxidative stress. After 20 min treatment at 100 ppm of hydrogen peroxide, it was 5 times higher than the control and 50 times higher than the control. The increase in viable cell number was confirmed. For 40 minutes after exposure to 500 ppm, the low-temperature pre-treated strains maintained high viable cell counts, and at 1,000 ppm, the viable cell viability decreased significantly in the control group without low-temperature pre-treatment. (See FIG. 4 ). The low temperature pretreatment effect on dry stress was confirmed that the number of viable cells of the low temperature pre-treated strain 100 times higher than the strain that was not pre-treated after 4 days (see Fig. 5 ).

In order to confirm the synthesis of new protein during low temperature pretreatment, the strain treated with chloramphenicol, a protein synthesis inhibitor, had no low temperature pretreatment effect, and it was confirmed that resistance enhancement effect was related to new protein synthesis at low temperature pretreatment (see FIG. 5 ). . In order to determine whether the csp gene, which is a cold shock related gene, is synthesized in the low temperature pretreatment process of Bradyrizium japonicum, PCR was confirmed, and it was confirmed that the csp gene was expressed during low temperature pretreatment (see FIG. 6 ). The amino acid sequence of SEQ ID NO: 4 of the csp showed high homology to the csp amino acid sequence of Escherichia coli and cynorizobium melorirot (see FIG. 7 ).

The effect of low temperature pretreatment on external stress can be explained by the change of membrane structure and the synthesis of CSP. The growth of the strain depends on the permeability maintained by the fluidity of the membrane and the composition and function of the lipids. At low temperatures, it is known that the proportion of lipids and the degree of desaturation increases, the length of residues decreases, and methyl residues increase. (Drouin et al., FEMS Microbiol. Ecol . 2000, 32: 111-120). In addition, new proteins are synthesized to survive at low temperatures, such as CSP synthesized at a low temperature for a certain time and CAP synthesized during low temperature culture (Panoff et al., Cryobiol. 1995, 32: 516-520). ). Expression of these proteins can be confirmed in several strains (Drouin et al., FEMS Microbiol. Ecol . 2000, 32: 111-120; Fujii et al., FEMS Microbiol Lett . 1999, 178: 123-128; Graumann et al. J. Bacteriol . 1996, 178: 4611-4619; Horton et al., Antonie van Leeuwenhoek , 2000, 77: 13-20). Most CSPs are less than 10 kDa in size, and in particular, the root-knot-producing strain Rhizobium leguminosarum synthesizes 6.1 kDa of CSP and is known to synthesize several proteins as CAPs (Drouin et al., FEMS Microbiol.Ecol. 2000, 32: 111-120).

As described above, since the low temperature pretreatment during the high temperature, drying, and oxidative stress processes acting as the main stress for the formulation showed a relatively high biocidal effect, the low temperature pretreatment method of the present invention is Bradyrizobium japoni. It can be usefully used for the preparation of microbial fertilizers using the strain by increasing the viability of the cum.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Low Temperature Pretreatment of Bradyrizium Japonicum

<1-1> Strains and Media Used

The present inventors examined the effect of low temperature pretreatment using the soil microorganism Bradyrizium japonicum 61A101c (Nitragin, Wiscosin). The microorganisms were incubated in AMA medium (mannitol 10 g; yeast extract 1 g; NaCl 0.2 g; K ₂ HPO ₄ 0.5 g; MgSO ₄ 0.2 g; FeSO ₄ 0.005 g; distilled water 1,000 ml). C, 150 rpm.

<1-2> Low Temperature Pretreatment of Bradyrizium Japonicum

The present inventors centrifuged the Bradyrizobium japonicum 61A101c for 16 hours (2 times the doubled time) at 4 ° C. and 28 ° C. at low temperature pretreatment conditions during the late growth phase or initial stagnation phase. Cells were harvested at (8,000 rpm, 15 minutes). After washing three times in 0.1 M PBS (phosphate buffered saline, pH 7.2), and resuspended so that OD ₆₀₀ (Optical Density) is 0.5 ~ 1.0 (CFU 0.5 ~ 1.0 × 10 ⁹ ) using 0.1 M PBS. The cells were subjected to various stress experiments. And colony counting was performed to measure the number of viable cells at every time exposed to stress, and the measured number of viable cells was expressed as viability (%) based on the initial number of colonies.

Example 2 Analysis of Resistance to Environmental Stress

The inventors have examined whether low temperature pretreated Bradyrizium japonicum is resistant to environmental stresses such as acids, heat, ethanol, salts and hydrogen peroxide ( FIG. 1 ).

<2-1> Effect of Low Temperature Pretreatment on High Temperature Stress

The present inventors have investigated the effect of resistance to the high temperature stress of the low temperature pretreatment Bradyrizium japonicum. Specifically, the cells were harvested by centrifugation (15,000 × g, 5 minutes) after diluting the cold pre-treatment and the low-temperature pre-treatment, resuspending in 0.1 M PBS (pH 7.2), and then 1 ml each in 1.5 ml microtubes. After dividing and incubating at 28 ℃ and 42 ℃ was measured the number of viable cells on a daily basis.

As a result, it was confirmed that the pretreatment when exposed to 42 ℃ to maintain more than 100 times the viability than that did not. When exposed to high temperature (42 ℃) for one day, the low temperature pre-treatment showed a reduction rate of 100 times or more than the initial cell number, it can be seen that the other is reduced by more than 1,000 times ( Fig. 2 ).

<2-2> Low Temperature Pretreatment Effect on Alcohol Stress

The present inventors have examined the resistance effect of the cold pretreated Bradyrizium japonicum to alcohol stress. Specifically, in order to confirm the effect of stress on alcohol, the strain was suspended in 5% or 10% ethanol solution and exposed for 0, 2, 4, 8 hours, and the number of viable cells was measured.

As a result, it was confirmed that about 1,000 times the viability was increased by the low temperature pretreatment effect. When exposed to 5% concentration, low temperature pretreatment decreased by 100 times up to 4 hours, otherwise decreased by 10,000 times. After 8 hours, both cases were killed. At 10% concentration, it can be confirmed that the viable cell number is maintained for 2 hours or more by pretreatment ( FIG. 3 ).

<2-3> Low Temperature Pretreatment Effect on Oxidative Stress

The present inventors have investigated the resistance effect of oxidative stress of low temperature pretreated Bradyrizium japonicum. Specifically, in order to confirm oxidative stress, 100, 500, and 1000 ppm of H ₂ O ₂ were added to 50 ml of the suspended strain, and the number of viable cells was measured after 0, 20, 40, or 60 minutes, respectively.

As a result, it was possible to confirm the low temperature pretreatment effect at each concentration. At 100 ppm of hydrogen peroxide concentration, after 20 minutes of treatment, the number of viable cells was increased by about 5 times and 40 times, but after 60 minutes, the pretreatment effect was not confirmed. For 40 minutes after exposure to 500 ppm, it was confirmed that the pretreated strain maintained a high viable count. At 1,000 ppm, non-pretreatment did not significantly reduce the viability, but the pretreated strain maintained the high viability for 20 minutes or more ( FIG. 4 ).

<2-4> Low Temperature Pretreatment Effect on Drying Stress

The present inventors have investigated the effect of resistance to dry stress of Bradyrizium japonicum pretreated at low temperature. Drying experiments were sterilized with 6 mm filter paper (Whatman, England) and inoculated with 5 μl of a suspension of strain each and dried for 1-4 days. The viable cell number was measured after 10 strains of filter paper inoculated with 1 ml of distilled water suspended in 1.5 ml microtube.

As a result, it was confirmed that the number of viable cells of the pretreated strain was about 100 times higher than the strain that was not pretreated after 4 days ( FIG. 5 ).

Example 3 Mechanism Analysis for Low Temperature Pretreatment Effect

In order to determine whether the resistance to environmental stress caused by low temperature pretreatment of Bradyrizium japonicum was attributable to the expression of intracellular genes, the present inventors examined the peptidyl-transferase of Chloramphenicol, which is known to inhibit protein synthesis by irreversibly binding to peptidyl-transferase), was studied.

Specifically, dry stress experiments were performed by pretreatment with low temperature pretreatment and pretreatment including chloramphenicol at a concentration of 50 μg / ml. Dry screed experiment was performed in the same manner as in Example <2-4>.

As a result, the low temperature pretreatment with chloramphenicol showed the same stress response as the low temperature pretreatment ( FIG. 5 ). This indicates that protein synthesis during cold pretreatment is essential for inducing resistance to stress.

Example 4 Gene Analysis Expressed in Low Temperature Pretreatment

In Example 3, the inventors of the present invention confirmed that protein synthesis is essential in the low temperature pretreatment, and performed a polymerase chain reaction (PCR) to identify genes expressed in the low temperature pretreatment.

<4-1> through PCR cspA Confirm

The inventors performed PCR analysis using primers containing conserved sites among several cspA gene sequences based on the expression of cspA gene during cold pretreatment in other microorganisms. Specifically, to separate chromosomal DNA (Sambrook et al., Molecular cloning , 2001, Cold Spring Harbor), PCR (Polymerase Chain Reaction) to perform CSP1 primers described in SEQ ID NO: 1 and CSP2 primers described in SEQ ID NO: 2 Used. The primers were prepared by analyzing conservative sequences in strains containing the csp gene (Panoff et al., Cryobiol , 1998, 36: 75-83). In order to clone the csp gene, each of the 5 ' Eco RI, Bam HI sites were included. To amplify the csp gene, the total amount of the PCR reaction was 25 μl, and 1 ng template DNA, 10 × reaction buffer, 10 mM dNTP mixture, 10 pmol primer (CSP1, CSP2) and 2.5 U Taq polymerase (TaKaRa, Japan) Amplify by mixing to concentration. In addition, E. coli JM109 was included as a control. PCR conditions were as follows; After 4 minutes of denaturation at 95 ° C., 15 seconds at 95 ° C., 30 seconds at 50 ° C., and 30 seconds at 72 ° C. were repeated 30 times, followed by further 4 minutes at 72 ° C. Amplified DNA was confirmed by electrophoresis in 1 × TAE buffer (pH 8.0) using 2.0% agarose gel containing ethidium bromide (0.5 μg / ml). 100 bp DNA ladder (TaKaRa, Japan) was used as a size marker.

As a result, the cold pretreatment-related gene used as a control, but less than the confirmed E. coli, but confirmed that the csp gene region of Bradyrizium japonicum amplified to a size of 200 bp ( Fig .

<4-2> Sequence Analysis

We analyzed the sequencing of Csp expressed during the cold pretreatment. An ABI PRISM 310 analyzer (Applied biosystems, USA) was used to identify the nucleotide sequence of the amplified csp gene, and the gene DOC program (Immunex Corporation, USA).

As a result, it was confirmed that the nucleotide sequence of the Csp gene is described by SEQ ID NO: 3 and the amino acid sequence inferred from the nucleotide sequence is described by SEQ ID NO: 4 .

Comparing the amino acid sequence with the amino acid sequence of the csp gene of E. coli and Sinorhizobium meliloti , which is already known , the gene of Bradyrizobium japonicum was isolated from other strains as well as from the other strains. The homology of the amino acid sequence was also shown with the gene ( FIG. 7 ).

As described above, the method of improving the bio-vibration by low-temperature pretreatment of the bradyrizobium japonicum of the present invention is to increase the resistance to stress received in the process of formulating the strain of the microbial fertilizer using the strain It can be usefully used for manufacturing.

<110> INHA UNIVERSITY <120> Method for improving viability performing cold adaptation of Bradyrhizobium japonicum <130> 2p-01-16 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CSP1 <400> 1 cccgaattcg gtacagtaaa atggttcaac gc 32 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> CSP2 <400> 2 cccggatccg gttacgttag cagctggcgg gcc 33 <210> 3 <211> 170 <212> DNA <213> Bradyrhizobium japonicum <400> 3 ccctgcaagg cttcggcttc atcactcctg acgatggctc taaagatgtg ttcgtacact 60 tctntgctat ccagaacgat ggttacaaat ctctggacga aggtcaggg cct <212> PRT <213> Bradyrhizobium japonicum <400> 4 Leu Gln Gly Phe Gly Phe Ile Thr Pro Asp Asp Gly Ser Lys Asp Val 1 5 10 15 Phe Val His Phe Xaa Ala Ile Gln Asn Asp Gly Tyr Lys Ser Leu Asp 20 25 30 Glu Gly Gln Lys Val Ser Phe Thr Ile Glu Ser Gly 35 40

Claims (3)

A method of enhancing the viability by low temperature pretreatment of Bradyrhizobium japonicum . The method of claim 1, wherein the pretreatment is a low temperature pretreatment at 4 to 15 ° C. The method of claim 1 wherein the pretreatment is a low temperature pretreatment for 6 to 24 hours.