CN112080546B

CN112080546B - Reverse screening method for acid-sensitive transposon insertion mutant

Info

Publication number: CN112080546B
Application number: CN202010951090.8A
Authority: CN
Inventors: 季学猛; 王硕
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2020-09-11
Filing date: 2020-09-11
Publication date: 2022-04-08
Anticipated expiration: 2040-09-11
Also published as: CN112080546A

Abstract

The invention provides a reverse screening method for acid-sensitive transposon insertion mutants, which comprises the following steps: (1) constructing a transposon insertion mutant library; (2) combined treatment of bactericidal antibiotics and acid; (3) culturing bacteria and picking single clone; (4) and (5) verifying acid-resistant phenotype of bacteria. The reverse screening method for the acid-sensitive transposon insertion mutant depends on the antagonism of the acidic environment to the bactericidal antibiotics, and the antibiotics can specifically kill replication active bacteria and have weaker bactericidal effect on bacteria with inhibited growth under the environmental stress. Test results show that the obtained mutant has the acid sensitivity rate as high as 100%, the method is simple and effective, the mutant is successfully applied to gram-negative representative bacteria, namely enterobacter sakazakii and gram-positive representative bacteria, namely listeria monocytogenes, and a new tool and a new method are provided for the research of gene functions.

Description

Reverse screening method for acid-sensitive transposon insertion mutant

Technical Field

The invention belongs to the field of gene screening, and particularly relates to a reverse screening method for an acid-sensitive transposon insertion mutant.

Background

Transposon (Tn), also called "transposon", refers to a DNA sequence that exists on chromosomal DNA and can replicate and displace autonomously. Transposons can be transferred between different replicons, inserted from one site to another in an abnormal recombination manner, and exert a variety of genetic effects on the structure and expression of the new site gene. Recent studies have demonstrated that transposons are ubiquitous in the biological world and are believed to play an important role in the genetic evolution of organisms. The process of transferring and excising a transposon from one position to another changes the structure and sequence of the original gene, thereby generating mutations.

Transposons have the function of causing random mutagenesis of DNA, are now one of the important means for studying gene function, and are widely applied to the construction of mutant libraries and gene targeting. Transposons have selectable resistance markers to facilitate identification of mutations in vivo, transposon-mutagenized mutants contain known DNA fragments, and the location of the mutated gene can be identified by crossing transposon sequences. Transposon-mutagenized mutants have a high degree of polarity, rendering the mutagenized gene and downstream genes within the same operon completely non-functional.

Transposon mutation can rapidly obtain a large number of random insertion mutants for the research purposes of identifying environmental tolerance genes, toxic genes, rate-limiting enzyme genes and the like. The selection of insertional mutations that lead to environmental tolerance is often simple and can be carried out in resistant media, environmentally tolerant strains having growth advantages being selected. However, sometimes wild-type bacteria are inherently resistant to the environment, and it is necessary to screen for environmental tolerance-determining genes, and to screen for environmentally sensitive insertion mutants and identify insertion sites, and in this case, it is necessary to screen for large numbers of clones.

Transposon mutation has been widely used in environmental tolerance and pathogenic mechanism research of pathogenic bacteria. The food-borne pathogenic bacteria need to pass through the acidic environment of the stomach and enter the intestinal tract to cause diseases. Generally, acid-sensitive bacteria are thought not to cause food-borne diseases, so that the tolerance to acidic environment is closely related to the pathogenic capability of food-borne pathogenic bacteria, and because of this, the mechanism of tolerance to acidic environment of food-borne pathogenic bacteria is receiving extensive attention and research. The mechanism of tolerance to acidic environments requires acquisition of mutants and phenotypic validation. Transposon mutagenesis, while rapidly producing large numbers of random mutants, results in less than one ten thousandth of acid sensitive mutants and requires extensive clonal screening and phenotypic validation. At present, no reported effective method for quickly and conveniently back-screening acid-sensitive strains exists.

Disclosure of Invention

In view of the above, the present invention is directed to overcoming the drawbacks of the prior art and providing a method for reverse screening of acid-sensitive transposon insertion mutants.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a reverse screening method for a transposon insertion mutant sensitive to acid, comprising the steps of:

(1) constructing a transposon insertion mutant library;

(2) combined treatment of bactericidal antibiotics and acid;

(3) culturing bacteria and picking single clone;

(4) and (5) verifying acid-resistant phenotype of bacteria.

Further, the bactericidal antibiotic in the step (2) is one of an antibiotic interfering cell wall synthesis or an antibiotic affecting bacterial protein synthesis; the concentration of the bactericidal antibiotic is 50-1000 mg/L.

Further, the antibiotic interfering the synthesis of the cell wall is one of beta-lactam antibiotics, vancomycin, teicoplanin or fosfomycin; the beta-lactam antibiotic is one of penicillin antibiotics or cephalosporins.

Further, the antibiotic affecting the synthesis of bacterial protein is an aminoglycoside antibiotic.

Further, the construction method of the transposon insertion mutant library in the step (1) is as follows: and amplifying the transposon, transferring the amplified transposon into bacteria for replication, and screening by bactericidal antibiotics to obtain the transposition mutator.

Further, the method for the combined treatment of the bactericidal antibiotic and the acid in the step (2) is as follows: the bacteria are cultured in an acid culture medium, and then the bactericidal antibiotics are added into the bacteria for continuous culture.

Further, the pH of the acidic medium is less than the optimum growth pH of the wild type of the bacteria; the pH value of the acidic culture medium is 1.5-3.0.

The application of the reverse screening method for the acid-sensitive transposon insertion mutant and the application of the method in reverse screening of recombinant bacteria.

The application of the reverse screening method for the acid-sensitive transposon insertion mutant and the application of the method in screening transformants.

The reverse screening method for the acid-sensitive transposon insertion mutant and the application of the method in screening strains which are subjected to spontaneous mutation or mutagen mutagenesis.

Transposon mutations can efficiently produce large numbers of near random mutations in the bacterial genome, however, subsequent screening for stress sensitive mutants typically requires extensive single colony screening since most transposon insertions do not result in the desired phenotypic differences. In order to quickly screen acid-sensitive mutants, the reverse screening method provided by the invention has antagonistic action on bactericidal antibiotics in an acid environment, and the antibiotics can specifically kill replication-active bacteria and have a weak bactericidal effect on bacteria with inhibited growth under environmental stress.

Compared with the prior art, the invention has the following advantages:

the reverse screening method for the acid-sensitive transposon insertion mutant depends on the antagonism of the acidic environment to the bactericidal antibiotics, and the antibiotics can specifically kill replication active bacteria and have weaker bactericidal effect on bacteria with inhibited growth under the environmental stress. Test results show that the obtained mutant has the acid sensitivity rate as high as 100%, the method is simple and effective, the mutant is successfully applied to gram-negative representative bacteria, namely enterobacter sakazakii and gram-positive representative bacteria, namely listeria monocytogenes, and a new tool and a new method are provided for the research of gene functions.

Drawings

FIG. 1 is a graph showing the killing effect of a bactericidal antibiotic according to example 1 on wild type Enterobacter sakazakii;

FIG. 2 is a bar graph showing the inhibition of growth of acid sensitive Enterobacter sakazakii by acid according to example 1 of the present invention;

FIG. 3 is a bar graph showing the results of a reverse screening of Enterobacter sakazakii mutants sensitive to acidity according to example 1 of the present invention;

FIG. 4 is a graph showing the growth of Enterobacter sakazakii mutant in acidic environment according to example 1 of the present invention: 4-A is sidA gene, 4-B is hmSP gene, 4-C is recA gene, 4-D is rpfR gene, 4-E is nlpD gene;

FIG. 5 is a graph depicting the killing of wild-type Listeria monocytogenes by the bactericidal antibiotic of example 2 of the present invention;

FIG. 6 is a bar graph of the growth inhibition of acid sensitive Listeria monocytogenes by the acid of example 2 of the present invention;

FIG. 7 is a bar graph of the results of a reverse screening of acid-sensitive Listeria monocytogenes mutants according to example 2 of the present invention;

FIG. 8 is a graph showing the growth of Listeria monocytogenes mutants according to example 2 of the present invention in an acidic environment: 8-A is lmo2027 gene, 8-B is lmo0051 gene, and 8-C is lmo1473 gene.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to examples.

Example 1

(1) bactericidal activity of gentamicin against enterobacter sakazakii (fig. 1): the gentamicin with the three concentrations has stronger bactericidal activity, and the active bacteria number of the enterobacter sakazakii is reduced to be below a detection threshold value within 60min by the gentamicin with the concentration of 200 mg/L;

(2) acid pH value: the growth inhibition effect of LB culture medium with different pH values regulated by hydrochloric acid on the Enterobacter sakazakii is that the dnaK gene deletion mutant strain of the Enterobacter sakazakii is taken as an acid-sensitive control bacterium, compared with neutral pH, the pH3.0 has obvious inhibition effect on the growth of delta dnaK, and has obvious inhibition effect on the growth of wild Enterobacter sakazakii (as shown in figure 2);

(3) construction of transposon insertion mutant pools: plasmid pUTimiTn 5 Km (Beijing Lebo Biotechnology Co., Ltd.) is transferred into E.coli lambda pir for amplification, then pUTimiTn 5 Km plasmid is extracted to prepare electric transformation competent Enterobacter sakazakii, and the pUTimiTn 5 Km plasmid is transferred into the Enterobacter sakazakii through electric transformation. The vector pUTimiTn 5 Km is used for leaving the kanamycin resistance gene in the Enterobacter sakazakii only when transposition occurs, and the culture medium is screened by kanamycin, so that the Enterobacter sakazakii which does not have transposition is killed, and cells containing the kana resistance gene are screened out. 5000 transposable mutants of Enterobacter sakazakii BAA894 are obtained by transposable mutagenesis;

(4) the survival rate of enterobacter sakazakii under the combined action of the acidic pH and the gentamicin is as follows: after the acid-sensitive delta dnaK mutant strain is inhibited from growing at an acidic pH, the acid-sensitive delta dnaK mutant strain can resist the killing effect of gentamicin (as shown in figure 3), on the contrary, the gentamicin has a quick killing effect on the acid-resistant wild strain, and the number of live bacteria of the acid-resistant wild strain is lower than a detection threshold after 60 minutes. Therefore, the results show that the combined application of the acidic pH and the gentamicin has a reverse screening effect on the acidic sensitive bacteria of the Enterobacter sakazakii;

(5) the screening effect of the combination of the acidity and the gentamicin on the enterobacter sakazakii transposon mutation acid sensitive bacteria is as follows: the transposon mutant bacteria solution is cultured in LB containing 50mg/L kanamycin for overnight, washed twice by hydrochloric acid acidified LB broth (pH3.0), 10mL of bacteria suspension with OD600 ═ 0.1 is prepared by LB broth (pH3.0), the bacteria suspension is incubated at 37 ℃ for half an hour, then gentamicin is added to make gentamicin in LB reach 100 mg/L concentration, the bacteria solution is cultured at 37 ℃ for 1 hour by shaking, then the strain is diluted and spread on LB agar (pH 7.0), LB agar plate is incubated overnight at 37 ℃ incubator, the monoclonal colony growing on LB agar is selected, and the acid sensitivity is determined by using the growth curve in acid;

(6) 5 monoclonals were obtained, and the insertion sites of the mutations were identified: the phenotype of acid sensitivity was further verified by knocking out the open reading frames of the genes, and finally, 5 genes, sidA, hmSP, recA, rpfR and nlpD, were identified, which enabled Enterobacter sakazakii to resist an acidic environment (as shown in FIG. 4), but did not affect the growth rate of the bacteria in neutral media.

Therefore, through reverse screening of a transposon mutation bacteria library of enterobacter sakazakii by combining acidic pH and gentamicin, 5 acid-sensitive transposon insertion mutation bacteria are efficiently obtained, and the bacteria are sensitive to acidic environment in the subsequent gene knockout process by confirming that 5 genes are knocked out. The success rate of the reverse screening method reaches 100 percent.

Gentamicin is a rapidly bactericidal aminoglycoside antibiotic, most effective against rapidly growing bacteria. Therefore, exposure to an acidic pH environment that inhibits the growth of acid-sensitive mutants can protect these strains from the bactericidal effect of gentamicin, while wild-type strains and mutants that can grow under acidic conditions are rapidly killed.

The genotypes of the strains and plasmids are shown in table 1.

TABLE 1 genotypes of strains and plasmids

Example 2

(1) bactericidal activity of gentamicin against listeria monocytogenes (see fig. 5): the gentamicin with the three concentrations has stronger bactericidal activity, and the live bacteria number of the listeria monocytogenes is reduced to be below a detection threshold value within 60min by the gentamicin with the concentration of 50 mg/L;

(2) acid pH value: the BHI culture medium with different pH values regulated by hydrochloric acid has an inhibition effect on the growth of Listeria monocytogenes, the recA gene deletion mutant strain of Listeria monocytogenes is used as an acid-sensitive control bacterium, and compared with neutral pH, pH3.0 has a remarkable inhibition effect on the growth of delta recA and has a remarkable inhibition effect on the growth of wild Listeria monocytogenes (as shown in figure 6);

(3) construction of transposon insertion mutant pools: plasmid pTV1OK containing transposon Tn917 is amplified in Bacillus subtilis PY143, then extracted, and an electrotransformation competent Listeria bacterium is prepared, the pTV1OK plasmid is transferred into the Listeria bacterium by electrotransformation, the Listeria bacterium without a Listeria replicon and an integration site on the pTV1OK vector is utilized, an erythromycin resistance gene can be remained in the Listeria bacterium only by transposition, the Listeria bacterium without transposition is killed by an erythromycin screening culture medium, and cells containing the kanamycin resistance gene are screened out. Obtaining 6000 Listeria monocytogenes transposable mutants by transposable mutagenesis;

(4) the reverse screening method of the antagonism of the acid pH value on the bactericidal activity of the gentamicin comprises the following steps: the survival rate of the listeria monocytogenes under the combined action of the acidic pH and the gentamicin is that the acid-sensitive delta recA mutant strain can tolerate the killing action of the gentamicin after inhibiting the growth at the acidic pH (as shown in figure 7), on the contrary, the gentamicin has a quick killing action on the acid-resistant wild strain, and the number of live bacteria of the wild strain and the number of live bacteria of the acid-sensitive strain have a significant difference (p is less than 0.05) when the acid-sensitive strain is treated for 60 minutes, so the result shows that the combined application of the acidic pH and the gentamicin has a reverse screening action on the acid-sensitive bacteria of the listeria monocytogenes;

(5) the screening effect of the combination of the acidity and gentamicin on the listeria monocytogenes transposon mutation acid-sensitive bacteria is as follows: the transposon mutant strain was cultured overnight in BHI containing 10mg/L erythromycin, and washed twice with HCl-acidified BHI broth (pH 3.0). Preparing 10mL of bacterial suspension with OD600 of 0.1 from BHI broth (pH3.0), incubating the bacterial suspension at 37 ℃ for half an hour, adding gentamicin to make the concentration of gentamicin in BHI reach 100 mg/L, shaking-culturing the bacterial suspension at 37 ℃ for 1 hour, diluting and spreading the bacterial strain on BHI agar (pH 7.0), incubating BHI agar plates in an incubator at 37 ℃ overnight, selecting monoclonal colonies growing on BHI agar, and determining the acid sensitivity by using an in-acid growth curve;

(6) 3 monoclonals were obtained, and the insertion sites of the mutations were identified: the acid sensitive phenotype was further verified by knocking out the open reading frames of the genes, and finally, 3 genes, lmo2027, lmo0051 and lmo1473, were identified, which enable listeria monocytogenes to withstand acidic environments (see fig. 8), but do not affect the growth rate of the bacteria in neutral media.

Therefore, through the combination of acidic pH and gentamicin, a transposon mutant strain library of the listeria monocytogenes is reversely screened, 3 acid-sensitive transposon insertion mutant strains are efficiently obtained, and the fact that the bacteria are sensitive to the acidic environment due to the knockout of 3 genes is confirmed in the subsequent gene knockout. The success rate of the reverse screening method reaches 100 percent.

The genotypes of the strains and plasmids are shown in table 2.

TABLE 2 genotypes of strains and plasmids

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for reverse screening of an insertion mutant of a acid-sensitive transposon, which comprises: the method comprises the following steps:

(1) constructing a transposon insertion mutant library;

(2) combined treatment of bactericidal antibiotics and acid;

(3) culturing bacteria and picking single clone;

(4) verifying acid-resistant phenotype of bacteria;

the transposon is plasmid pUTimiTn 5 Km;

the method for combined treatment of the bactericidal antibiotics and the acid in the step (2) comprises the following steps: culturing bacteria in an acidic culture medium, and adding a bactericidal antibiotic into the bacteria for continuous culture; the pH of the acidic medium is less than the optimum growth pH of the wild type of the bacteria; the pH value of the acidic culture medium is 1.5-3.0;

the bactericidal antibiotic is gentamicin; the concentration of the bactericidal antibiotic is limited to 50 mg/L.

2. The method of claim 1 for reverse screening of acid-sensitive transposon insertion mutants, wherein: the construction method of the transposon insertion mutant library in the step (1) is as follows: and amplifying the transposon, transferring the amplified transposon into bacteria for replication, and screening by bactericidal antibiotics to obtain the transposition mutator.

3. Use of the method for reverse screening of acid-sensitive transposon insertion mutants of claim 1 or 2, wherein: the method is applied to reverse screening of recombinant bacteria.

4. Use of the method for reverse screening of acid-sensitive transposon insertion mutants of claim 1 or 2, wherein: the method is applied to screening transformants.

5. Use of the method for reverse screening of acid-sensitive transposon insertion mutants of claim 1 or 2, wherein: the use of said method for screening strains which are mutagenized by spontaneous mutation or by mutagens.