CN101302482B - Marine streptomyces S187 having wide-spectrum antibacterial activity - Google Patents
Marine streptomyces S187 having wide-spectrum antibacterial activity Download PDFInfo
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Abstract
The invention relates to marine streptomyces S187 having broad-spectrum antibacterial activity, belonging to the microbiological actinomycetes field. The marine streptomyces S187 is separated from a sea mud sample at a depth of 20 meters underwater in coastal waters. Primary screening through an agar block method proves that the bacterial strain has very strong antibacterial activity to Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger; the result of fermentation liquid secondary screening confirms good activity for antagonizing the Escherichia coli, the Staphylococcus aureus and the Bacillus subtilis. The analysis for 16S rDNA shows that the only actinomycetes having the homology as high as 99 percent with the marine streptomyces S187 is the salt lake bacterial strain Streptomyces chungwhensis (DSM41483) separated by Korean scholars, but the bacterial strain is separated from the sea mud sample for the first time. I type polyketide synthase (PKSI) is successfully amplified out from the streptomyces S187, which indicates that the streptomyces S187 has the potential ability of generating polyketide.
Description
Technical field
The present invention relates to the marine streptomyces S187 that a strain has broad spectrum antibiotic activity, it belongs to microbiological actinomycetes field.
Background technology
Actinomycetes are gram positive bacteriums of class height (G+C) %, because its ability that has unique synthetic multiple baroque secondary metabolite has caused people's extensive concern.Many actinomycetic secondary metabolites have the purposes of medicine and plant protection aspect, have been widely used as antibacterium, antimycotic and antitumor drug.In the tens thousand of kinds of microbe-derived biologically active substances of now having found, have 70% to be approximately by actinomycetes institute synthetic.
Yet along with the minimizing of finding new natural active matter from soil actinomycete, add the continuous generation of resistant bacterium, the marine actinomycete resource becomes the new source of finding new drug.The ocean account for earth surface long-pending 71%, the Microbial resources that wherein are richly stored with, the ocean environment of special complexity (high salinity, high pressure, low temperature and special illumination etc.) is given marine microorganism and is produced the special outcome that is different from the land microorganism.In the extreme environment of these so-called life, marine actinomycete has been developed and unique metabolic way, and this guarantees that not only it survives in extreme environment, the potentiality that produce antibiotic compounds also are provided.Therefore, ocean environment will become the important new source of actinomycetes and actinomycetes meta-bolites.
Summary of the invention
In order to solve the problem that existing actinomycetes field exists, the present invention utilizes the marine microorganism resource, provide a strain to have the marine streptomyces of broad spectrum antibiotic activity, this marine streptomyces is to staphylococcus, intestinal bacteria, Pseudomonas aeruginosa, white candiyeast, aspergillus niger and subtilis etc. all has good antibacterial activity.
A strain involved in the present invention has marine streptomyces (the Latin classification name: Streptomyces of broad spectrum antibiotic activity, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, depositary institution address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, CGMCC number: 2251, preservation date: on November 12nd, 2007) separate in the ooze sample, pretreatment process is for disperseing and differential centrifugation (DDC), isolation medium is oligotrophic substratum M5, and the purifying substratum is Bennett
Substratum, marine streptomyces S187 have the spore of xanchromatic substrate mycelium and aerial hyphae and white, do not produce soluble pigment.
Marine streptomyces S187 has broad spectrum antibiotic activity, to intestinal bacteria, and streptococcus aureus, subtilis, Pseudomonas aeruginosa, white candiyeast and aspergillus niger all have anti-microbial activity.
Marine streptomyces S187 amplifies I type polyketide synthase (PKSI) gene from its genome, and can produce polyketide.Provide the foundation for further new microbiotic being created in its other bioactive research unit and biosynthesizing.
Above-mentioned marine streptomyces S187 be from offshore waters following 20 meters obtain the ooze sample, adopt to disperse to isolate a strain marine streptomyces S187 with differential centrifugation (DDC) pretreatment process and specific oligotrophic isolation medium (M5).Determine its kind classification by evaluation in conjunction with physiological and biochemical test, the antibacterial tests of six kinds of pattern bacterium is determined its anti-microbial activity, the amplification of its polyketide synthase I (PKSI) gene is determined that it produces the potentiality of polyketide 16S rDNA.
Adopt the method for disperseing, can break up the particle of assembling in the ooze sample effectively, actinomycetes and spore thereof can be discharged effectively with differential centrifugation; Use oligotrophic substratum M5 (only containing agar and seawater), can avoid bacterium and other actinomycetic interference of growth fast, this is consistent with the oligotrophic condition of ocean environment.
The invention has the beneficial effects as follows:
(1) marine streptomyces S187 has broad spectrum antibiotic activity, to the strains tested streptococcus aureus, and intestinal bacteria, Pseudomonas aeruginosa, white candiyeast, aspergillus niger and subtilis all have the excellent antibiotic activity.
(2) polyketide synthase I (PKSI) gene that amplifies from marine streptomyces S187 has disclosed it and has produced the potentiality of polyketide.
Embodiment
Fig. 1 shows the position of S187 in the phylogenetic tree that makes up.
One, separating marine streptomycete S 1-5 87 and as follows to its job step of studying:
The first step: the separation of bacterial classification
Between in August, 2006 from Xiao Pingdao marine site, Dalian under water 20 meters obtain the ooze sample, sample retention is in ice chest and take back the laboratory immediately and handle.Sample adopts the method for disperseing with differential centrifugation (DDC), adopts oligotrophic substratum M5 as isolation medium, cultivates a week down in 28 ℃, separates obtaining marine streptomyces S187, adopts the Bennett substratum as the purifying substratum.
Second step: anti-microbial activity
Anti-microbial activity is measured substratum: bacterium is used nutrient broth agar, and white candiyeast is a wort agar, and mould is adopted Czapek's agar.Bacterium was in 37 ℃ of cultivations 24 hours, and fungi was cultivated 48 hours down in 32 ℃, adopted the multiple sieve method of agar block method primary dcreening operation and fermented liquid to carry out anti-microbial activity mensuration, and the size of record inhibition zone.Strains tested streptococcus aureus (1.89), intestinal bacteria (1.797), Pseudomonas aeruginosa (1.2031), white candiyeast (2.538), aspergillus niger (3.2915) is available from Chinese common micro-organisms culture presevation administrative center (in the bracket is bacterium numbering), and subtilis is given by professor Xu Zhouyuan of Korea S Myongji University.
The 3rd step: strain identification
Extract marine streptomyces S187 genomic dna, adopt bacterium universal primer amplification 16S rDNA and purifying order-checking, sequencing result carries out homology relatively by the Blastn program of NCBI, adopts MEGA3.1 software, sets up phylogenetic tree with the Neighbor-Joining method.The cultural characteristic of bacterial strain and physiological and biochemical property carry out according to classification of actinomycetes and identification handbook.By sequential analysis, find that it is Streptomyces chungwhensis (DSM4l 843) that unique and marine streptomyces S187 have the streptomycete of 99% homology, and S.chungwhensis separates from the Korea S salt lake, except 16S rDNA sequence, does not have other relevant research reports.
The form comparison shows that in conjunction with physiological and biochemical test S187 and S.chungwhensis have very big difference, infers that marine streptomyces S187 is the ocean mutation of S.chungwhensis.
The 4th step: the amplification of polyketide synthase I (PKSI) gene.
Utilize degenerate primer:
5 '-CCSCAGSAGCGCSTSCTSCTSGA-3 '/5 '-GTSCCSGTSCCGTGSGCCTCSA-3 ' increases to the polyketide synthase gene, successfully obtain positive fragment, carry out transformation experiment and order-checking, sequence is submitted to GenBank obtains sequence number EU057563.
Two, marine streptomyces S187 adopts the agar block method antibacterial experiment:
The first step: the cultivation of actinomycetes and strains tested
Marine streptomyces S187 is cultivated a week under 28 ℃ in the Bennett substratum, and visible thalline generates white spore.Get the agar block that contains spore and substrate mycelia and in the TSB substratum, carry out liquid culture, for examination sensitive bacterial nutrient broth medium, the white candiyeast is a malt extract medium, mould is adopted czapek's solution, bacterium was cultivated 24 hours in 37 ℃, fungi was cultivated 48 hours down in 32 ℃, and rotating speed is 150 rev/mins of cultivations.
Second step: will be inoculated in the corresponding separately substratum for the examination sensitive strain, method is: the solid medium heating is dissolved, add the corresponding strains tested of 100 microlitres when being cooled to 60 ℃ of left and right sides, mixing is cooled to culture medium solidifying.
The 3rd step: take off cylindrical cultured actinomycetes with 1ml rifle head, be inverted in the substratum that has solidified.Bacterium was in 37 ℃ of cultivations 24 hours, and fungi was cultivated 48 hours down in 32 ℃.Observe the size of inhibition zone.
Following table has been listed the antibacterial circle diameter (millimeter) of marine streptomyces S187 employing agar block method primary dcreening operation.
Three, marine streptomyces S187 adopts the broth method antibacterial experiment:
The first step: the cultivation of actinomycetes and strains tested
Marine streptomyces S187 is cultivated a week under 28 ℃ in the TSB liquid nutrient medium, and rotating speed is 150 rev/mins.Will be for examination sensitive strain liquid culture, bacterium is used nutrient broth medium, and white candiyeast is a malt extract medium, and mould is adopted czapek's solution, and bacterium was in 37 ℃ of cultivations 24 hours, and fungi was cultivated 48 hours down in 32 ℃, and rotating speed is 150 rev/mins of cultivations.
Second step: strains tested is inoculated in the corresponding separately substratum, and method is: the solid medium heating is dissolved, add the corresponding strains tested of 100 microlitres when being cooled to 60 ℃ of left and right sides, mixing is cooled to culture medium solidifying.
The 3rd step: beat cylindrical hole with 1ml rifle head at the substratum that has solidified, collect actinomycetes fermentation liquor, 10000 rev/mins centrifugal 5 minutes, get in the supernatant liquor 300 microlitres adding cylindrical hole.Bacterium was in 37 ℃ of cultivations 24 hours, and fungi was cultivated 48 hours down in 32 ℃.Observe the size of inhibition zone.
Fermented liquid antibacterial experiment result:
Four, polyketide synthases sequence and the analysis of marine streptomyces S 187
From S187, successfully amplify I type polyketide synthase gene PKS187, carrying out sequential analysis by blastP finds, PKS187 has higher homology with the important antibiotic PKS gene fragment in a lot of streptomycetes source, these microbiotic comprise various antimycotic, anti-bacterial drugs, antitubercular agent, and antitumor drug.With its homology the highest be the synthetic polyketide synthases of latest find with microbiotic meridamycin of neurotization function, confirmed that further it produces the possibility of novel polyketides.The result of these sequential analyses provides clue for other physiologically active of further analyzing marine streptomyces S187.Growing tree (Fig. 1) by constructing system shows, PKS 187 and EpoD (P.cellulosum AAF62883), AbmE (P.cellulosum ABK32259) and JerD (P.cellulosum ABK32290) are in a branch, and it is far away with the PKSI fragment evolutionary distance in other streptomycete source, the novelty that has shown this sequence is for the horizontal transfer of further studying this section PKSI gene is laid a good foundation.This is studied isolating sequence and also lays the foundation for screening later on complete PKSI gene cluster and further combination biosynthesizing.
Five, the comparison of marine streptomyces S 187 and Streptomyces chungwhensis
Compared marine streptomyces S187 and the Streptomyces chungwhensis difference aspect anti-microbial activity, morphological character and physiological and biochemical property below respectively, the result shows, there are significant difference in marine streptomyces S187 and Streptomyces chungwhensis, and marine streptomyces S187 is a new streptomycete bacterial strain.
1, marine streptomyces S187 has the excellent antibiotic activity to six kinds of type strains, and Streptomyceschungwhensis does not all have activity to six kinds of pattern bacterium.
2, marine streptomyces S187 and the S.chungwhensis cultural characteristic on the Bennett substratum is shown in following table.According to morphologic observation, marine streptomyces S187 is a streptomycete.After cultivating for two weeks on the Bennett substratum, marine streptomyces S187 has the spore of xanchromatic substrate mycelium and aerial hyphae and white, does not produce pigment; And S.chungwhensis has the substrate mycelium of brown, and the spore of gray aerial hyphae and white produces, and produces melanochrome.Aspect salt tolerance, marine streptomyces S187 can only contain well-grown on the Bennett substratum of 0%-6%NaCl, containing on 9% and 12% the Bennett substratum, the growth of marine streptomyces S187 is suppressed, and S.chungwhensis well-grown on the substratum of the Bennett that contains 0%-12%NaCl.
3, following table is the physiological and biochemical test result of marine streptomyces S187 and S.chungwhensis.In four kinds of Physiology and biochemistries experiment of being done, marine streptomyces S187 and S.chungwhensis have 2 significantly differences, and marine streptomyces S187 can not hydrolyzed starch, but can produce H
2S; S.chungwhensis energy hydrolyzed starch, but can not produce H
2S.In the sole carbon source experiment, marine streptomyces S187 can better utilize glucose than S.chungwhensis, D-seminose and rhamnosyl, but wood sugar and N.F,USP MANNITOL can not be utilized, but S.chungwhensis can utilize all sole carbon source growths of using in the experiment, can be utilized best as sole carbon source by marine streptomyces S187 be rhamnosyl and acetate, glucose, sucrose, sorbyl alcohol, fructose, D-seminose and raffinose take second place, and on the contrary, it is best that S.chungwhensis grows on gluconate and fructose, at sucrose, maltose, inositol, sorbyl alcohol, N.F,USP MANNITOL, growth is taken second place on raffinose and the acetate.Marine streptomyces S187 better than time growth in the time of 28 ℃ at 20 ℃, and marine streptomyces S187 is fine 28 ℃ and 20 ℃ of growths, shows that marine streptomyces S187 has better tolerance to low temperature, this is consistent with its isolating environment from ooze.
Claims (1)
1. a strain has the marine streptomyces S187 (Streptomyces) of broad spectrum antibiotic activity, it is characterized in that: registering on the books of described marine streptomyces S187 is numbered CGMCC No.2251, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on November 12nd, 2007.
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| CN101942425B (en) * | 2010-08-24 | 2012-06-20 | 北京农学院 | Enzyme related to benzalacetone synthesis, coding gene and application thereof |
| CN102220271B (en) * | 2011-05-27 | 2012-12-19 | 中国科学院微生物研究所 | Marine Streptomyces strain and use thereof |
| CN102268392B (en) * | 2011-07-12 | 2013-01-23 | 中国科学院新疆生态与地理研究所 | Method for separating and screening oligographic bacterium producing protease |
| CN102703362B (en) * | 2012-06-21 | 2013-06-05 | 福建农林大学 | Streptomyces fujianensis sp and DNA identification method |
| CN103789221B (en) * | 2013-04-28 | 2016-05-04 | 北京农学院 | There is actinomyces and the screening technique thereof of broad-spectrum antibacterial activity |
| CN107299064B (en) * | 2016-09-29 | 2020-02-18 | 天津大学 | Marine actinomycete S-19-3 with broad-spectrum antibacterial activity |
| CN107164267B (en) * | 2017-06-05 | 2020-03-17 | 天津大学 | Marine actinomycete U-19-3 with broad-spectrum antibacterial activity and application thereof |
| CN107699526B (en) * | 2017-11-20 | 2021-05-25 | 运城学院 | Actinomycete strain for preventing and treating gray mold and application thereof |
| CN108504592B (en) * | 2018-03-26 | 2021-01-05 | 曲阜师范大学 | Marine actinomycete and screening and application thereof |
| CN108753663A (en) * | 2018-06-28 | 2018-11-06 | 四川大学 | One plant of streptomycete and its application with antibacterial activity |
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