微生物生理與代謝調控 3 20130320

1、微生物生理與代謝調控,Microbial Physiology and Metabolic Regulation,趙心清,大連理工大學生命科學與技術學院,2013.3.20,調課通知,3.27-,調至,4.6 (,星期六下午,1, 2,節(jié),),3.29-,調至,4.13 (,星期六下午,1, 2,節(jié),),4.10-,調至,4.20 (,星期六下午,1, 2,節(jié),),給大家造成的不便非常抱歉！,Industrial microorganisms,?,Bacillus,?,Lactobacillus,?,Corynebacteria,?,Actinomycetes,?,Yeast,?,Molds,

2、?,Microalgae,Important points of applied microbiology,?,Physiology,?,Biochemistry and,Genetics,?,Engineering,?,Ecology,Chapter 2,Bacillus,?,Biological aspects,?,Commonly used species,?,Extremophiles of,Bacillus,species,?,Utilization of,Bacillus,species,?,Genetics manipulation,Biological aspects of,B

3、acillus spp.,?,Gram positive,?,Endospore forming,?,Genetically amentable.,Endospore of,Bacillus subtilis,Thermotolerant, alkali tolerant,Common,Bacillus,species,?,Bacillus licheniformis,(,地衣芽孢桿菌）,?,Bacillus cereus,(,蠟樣芽孢桿菌,)*,?,Bacillus subtilis,(,枯草芽孢桿菌）,?,Bacillus thuringiensis,(,蘇云金芽孢桿菌）,?,Comple

4、te genomic DNA sequence of Bacillus,subtilis was released (in the year 1997, total,4.2 Mbp) and 4100 genes were identified,Kunst, Ogasawara, Moszer, Albertini, Alloni,Azevedo, Bertero, Bessieres, Bolotin, Borchert,S. et al. (1997) Nature 90, 249-256.,Extremophiles of,Bacillus,spp.,?,Thermotolerant,B

5、acillus,spp.,?,Alkali tolerant,B. alcalophilus,(pH 11).,B. firmus.,?,Organic solvent-tolerant,Bacillus,. (,B. subtilis,AHU1219),Utilization,of,Bacillus,spieces,?,Enzymes,protease, lipase, amylase,，,cellulase,pectinase,?,Polymers,hyaluronic acid, poly-gamma glutamic acid,?,Antibiotics,non-rebisomal p

6、eptide antibiotics,aminoglycoside antibiotics, polymyxins.,biocontrol, pesticides,?,microeclogial modulator,Production of hyaluronic acid by genetically,engineered,Bacillus subtilis,in Novozymes,Market: $ 500 million for HA,containing medical products, $,25 million for medical use.,?,Bioresour Techn

7、ol.,2013 Mar;132:427-31.,doi: 10.1016/j.biortech.2012.12.150. Epub,2013 Jan 3.,?,Metabolic engineering of,Bacillus,subtilis,for the efficient biosynthesis of,uniform hyaluronic acid with controlled,molecular weights.,?,Jia Y,Zhu J,Chen X,Tang D,Su D,Yao,W,Gao X,.,?,State Key Laboratory of Natural Me

8、dicines, School of,Life Science and Technology, China Pharmaceutical,University, Nanjing 210009, PR China.,?,Abstract,?,Bacillus subtilis,was engineered into an,efficient hyaluronic acid (HA) producer by,introducing two inducible artificial operons,carrying HA synthase gene from,Pasteurella multocid

9、a,多殺巴斯德菌,and,precursor genes encoding enzymes,involved in synthesis of the sugar,precursors.,?,A two-stage induction strategy was,established for metabolic engineering of,recombinant,B. subtilis,to efficiently,produce uniform HA with controlled,molecular weights.,?,Strain TPG223 produced larger HA,m

10、olecules (yield=6.8g/L; molecular,weight=4.5MDa) than strain PG6181,(yield=2.4g/L; molecular weight=13KDa),indicating that the enzymes involved in the,synthesis of UDP-glucuronic acid UDP-,葡,萄醛酸,are essential for HA biosynthesis.,?,Strain TPG223 was able to synthesize HA,molecules ranging in molecul

11、ar weight,from 8 KDa to 5.4 MDa indicating that size,control is achievable in vivo through,appropriate tools. The work reported here,not only advanced mechanisms research,of size control in vivo, but also could be an,attractive alternative for commercial,preparation of uniform size-defined HA.,?,J I

12、nd Microbiol Biotechnol.,2013 Mar 15.,Epub ahead of print,?,Purification, characterization and,cloning of a thermotolerant isoamylase,produced from,Bacillus sp,. CICIM 304.,?,Li Y,Niu D,Zhang L,Wang Z,Shi G,.,?,Source,?,Research Center of Bioresource 158(Pt 12):3072-82. doi:,10.1099/mic.0.063131-0.

13、Epub 2012 Oct 25.,?,Direct surfactin-gramicidin S,antagonism supports detoxification in,mixed producer cultures of Bacillus,subtilis and Aneurinibacillus,migulanus.,?,Rautenbach M,Ey,gh,-Bickong HA,Vlok NM,Stander,M,de Beer A,.,?,Source,?,BIOPEP Peptide Group, Department of Biochemistry,University o

14、f Stellenbosch, Matieland, South Africa.,?,Abstract,?,Antibiotic production as a defence,mechanism is a characteristic of a wide,variety of organisms. In natural,evolutionary adaptation, cellular events,such as sporulation, biofilm formation and,resistance to antibiotics enable some,micro-organisms

15、to survive environmental,and antibiotic stress conditions.,?,The two antimicrobial cyclic peptides in,this study, gramicidin S (GS),短桿菌肽,from,Aneurinibacillus migulanus,and the,lipopeptide surfactin,表面活性素,(Srf) from,Bacillus subtilis, have been shown to,affect both membrane and intercellular,compone

16、nts of target organisms. Many,functions, other than that of antimicrobial,activity, have been assigned to Srf.,?,We present evidence that an additional,function may exist for Srf, namely that of a,detoxifying agent that protects its producer,from the lytic activity of GS. We observed,that Srf produc

17、ers were more resistant to,GS and could be co-cultured with the GS,producer. Furthermore, exogenous Srf,antagonized the activity of GS against,both Srf-producing and non-producing,bacterial strains.,?,A molecular interaction between the,anionic Srf and the cationic GS was,observed with circular dich

18、roism and,electrospray MS. Our results indicate that,the formation of an inactive complex,between GS and Srf supports resistance,towards GS, with the anionic Srf forming a,chemical barrier to protect its producer.,This direct detoxification combined with,the induction of protective stress,responses

19、in,B. subtilis,by Srf confers,resistance toward GS from,A. migulanus,and allows survival in mixed cultures.,?,PLoS One.,2011;6(7):e22389. doi:,10.1371/journal.pone.0022389. Epub 2011 Jul 21.,?,Expression of the lantibiotic mersacidin,in Bacillus amyloliquefaciens FZB42.,?,Herzner AM,Dischinger J,Sze

20、kat C,Josten M,Schmitz S,Yak,l,ba A,Reinartz R,Jansen A,Sahl HG,Piel J,Bierbaum G,.,?,Source,?,Institute of Medical Microbiology, Immunology and,Parasitology, University of Bonn, Bonn, Germany.,?,Abstract,?,Lantibiotics are small peptide antibiotics,that contain the characteristic thioether,amino ac

21、ids lanthionine and,methyllanthionine.,?,As ribosomally synthesized peptides,lantibiotics possess biosynthetic gene,clusters which contain the structural gene,(lanA) as well as the other genes which,are involved in lantibiotic modification,(lanM, lanB, lanC, lanP), regulation (lanR,lanK), export (la

22、nT(P) and immunity,(lanEFG).,?,The lantibiotic mersacidin is produced by,Bacillus sp,. HIL Y-85,54728, which is not,naturally competent.,METHODOLOGY/PRINCIPAL FINDINGS:,The aim of these studies was to test if the,production of mersacidin could be,transferred to a naturally competent,Bacillus,strain

23、employing genomic DNA of,the producer strain.,?,Bacillus amyloliquefaciens,FZB42 was,chosen for these experiments because it,already harbors the mersacidin immunity,genes. After transfer of the biosynthetic,part of the gene cluster by competence,transformation, production of active,mersacidin was ob

24、tained from a plasmid in,trans.,?,Furthermore, comparison of several DNA,sequences and biochemical testing of,B.,amyloliquefaciens,FZB42 and,B. sp,. HIL,Y-85,54728 showed that the producer,strain of mersacidin is a member of the,species,B. amyloliquefaciens,.,?,CONCLUSIONS/SIGNIFICANCE: The,lantibio

25、tic mersacidin can be produced in,B. amyloliquefaciens,FZB42, which is,closely related to the wild type producer,strain of mersacidin. The new mersacidin,producer strain enables us to use the full,potential of the biosynthetic gene cluster,for genetic manipulation and downstream,modification approac

26、hes.,Food and cosmetics industries as well as medicine: thickener,humectant, bitterness relieving agent, drug carrier,biodegradable fibers, heavy metal absorber,Chapter 3. Lactic acid bacteria,乳酸菌,Diverse genus of lactic acid bacteria,Lactobacillus,(,乳酸桿菌屬,),，,Bifidobacterium,(,雙歧,桿菌屬,),Streptococcu

27、s,(,鏈球菌屬,),、,Leuconostoc,(,明串珠球菌屬,),、,Enterococcus,(,腸,球菌屬,),、,Lactococcus,(,乳球菌屬,),、,Carnobacterium,(,肉食桿菌屬,),、,Atopobium,(,奇,異菌屬,),、,Pediococcus,(,片球菌屬,),、,Aerococcus,(,氣球菌屬,),、,Vagococcus,(,漫游球菌,屬,),、,Listeria,(,李斯特氏菌屬,),、,Sporolactobacilus,屬,(,芽孢乳桿菌,),、,Bacillus,(,芽,孢桿菌屬少數(shù)種,),、,Brochothrix,(,環(huán)絲菌

28、屬,),、,Erysipelothrix (,丹毒絲菌屬,),、,Gemella,(,孿生菌屬,),和,Saccharococcus,(,糖球菌屬,),。,Feed, microecological modulator,Generally are chained cocci or rod,shaped,gram-positive, nonmotile,nonsporulating bacteria that produce,lactic acid,as a major or sole,product,of,fermentative metabolism and use,lactose as t

29、heir main source of carbon to,produce energy.,Biological aspects,“Kimchi” and SARS,2003,Identification of lactic acid bacteria,isolated from kimchi and studies on,their suitability for application as,starter culture in the production of,fermented sausages,Meat science,2006,vol.,72,no3,pp.,437-,445,?

30、,A total of 31 strains of lactobacilli,were,isolated from kimchi on the 4-6th day of,fermentation at 20,o,C using,MRS agar,plates,and identified on the basis of morphological,biochemical, and physiological,characteristics.,?,As a result only,L. plantarum,(,植物乳桿菌,),among the isolates from kimchi had

31、an ability,to adapt to the complex environment of,fermented sausage,which will thereby allow,them to act as starter cultures and natural,preservatives in sausage production.,?,Gut. 2011 Apr 6. Epub ahead of print (IF 9.3),?,Anti-inflammatory capacity of selected lactobacilli in,experimental colitis

32、is driven by NOD2-mediated,recognition of a specific peptidoglycan-derived,muropeptide.,?,Background and aims,Inflammatory bowel,disease (IBD),炎癥性腸病,has been linked to a,loss of tolerance towards the resident microflora.,Therapeutic use of probiotics is known to be,strain specific, but precise mecha

33、nisms remain,unclear. The role of NOD2 signalling and the,protective effect of,Lactobacillus,peptidoglycan,肽聚糖,(PGN) and derived muropeptides,胞壁肽,in experimental colitis were evaluated.,?,Methods,The anti-inflammatory capacity of,lactobacilli and derived bacterial,compounds was evaluated using the,2

34、,4,6-trinitrobenzene sulfonic acid (TNBS),colitis model. The role of NOD2, MyD88,and interleukin 10 (IL-10) in this protection,was studied using Nod2(-/-), MyD88(-/-),and Il10-deficient mice, while induction of,regulatory dendritic cells (DCs) was,monitored through the expansion of,CD103(+) DCs in m

35、esenteric lymph nodes,or after adoptive transfer of bone marrow-,derived DCs.,?,The development of regulatory T cells was investigated,by following the expansion of CD4(+)FoxP3(+) cells.,High-performance liquid chromatography and mass,spectrometry were used to analyse the PGN structural,differences.

36、,?,Results,The protective capacity of strain,Lactobacillus,salivarius,Ls33 was correlated with a local IL-10,production and was abolished in Nod2-deficient mice.,PGN purified from Ls33 rescued mice from colitis in an,IL-10-dependent manner and favoured the development,of CD103(+) DCs and CD4(+)Foxp3

37、(+) regulatory T cells.,In vitro Ls33 PGN induced IL-10-producing DCs able to,achieve in vivo protection after adoptive transfer in a,NOD2-dependent way.,?,This,protection,was,also,correlated,with,an,upregulation,of,the,indoleamine,2,3-dioxygenase,immunosuppressive,pathway,.,The,protective,capacity,

38、was,not,obtained,with,PGN,purified,from,a,non-anti-inflammatory,strain.,Structural,analysis,of,PGNs,highlighted,in,Ls33,the,presence,of,an,additional,muropeptide,M-tri-Lys.,The,synthesised,ligand,protected,mice,from,colitis,in,a,NOD2-dependent,but,MyD88-,independent,manner.,Conclusions,The,results,i

39、ndicated,that,PGN,and,derived,muropeptides,are,active,compounds,in,probiotic,functionality,and,might,represent,a,useful,therapeutic,strategy,in,IBD.,?,J Food Prot. 2011 Apr;74(4):651-7.,?,Purification and Characterization of Antifungal,-,Dodecalactone (,-,十二內酯,) from,Lactobacillus,plantarum,AF1 Isol

40、ated from Kimchi.,?,The aim of this study was to purify and identify an,antifungal compound from,Lactobacillus plantarum,AF1, which was isolated from kimchi. The antifungal,compound was purified by solid-phase extraction and,recycling preparative high-performance liquid,chromatography, and its struc

41、ture was elucidated by,using gas chromatography-mass spectrometry (GC-,MS).,?,The active compound from,L. plantarum,AF1 was,confirmed to be,-dodecalactone (molecular weight,198.3) by comparison of its gas chromatographic,retention time with the mass spectrum of standard,-dodecalactone. The MICs of,-

42、dodecalactone,against various fungi and bacteria ranged from 350,to 6,250 mg/ml.,-Dodecalactone showed strong,antifungal activity against molds,Aspergillus flavus,A. fumigatus,A. petrakii,A. ochraceus,A. nidulans,and,Penicillium roqueforti,.,?,The three tested yeast strains of,Candida,albicans,were

43、more resistant than the molds.,Antibacterial activity was evident but less potent,than the antifungal activity.,-Dodecalactone,produced pleasurable (fruity) organoleptic (,器官,感覺的,) characteristics. The results indicate the,potential of the,-dodecalactone produced by,L.,plantarum,AF1 as a biopreserva

44、tive and,flavoring compound, as well as a biosafe,remedy for candidiasis.,?,植物乳桿菌（,Lactobacillus plantarum,）是,乳酸桿菌中的一種，常存在于發(fā)酵的蔬菜,和果汁中。植物乳桿菌作為人體胃腸道的,益生菌群，具有,維持腸道內菌群平衡、提,高機體免疫力和促進營養(yǎng)物質吸收,等多種,功能。植物乳桿菌在食品工業(yè)中應用十分,廣泛。由于對食鹽、亞硝酸鹽具有良好的,耐受性，對蛋白質、脂肪無明顯直接的分,解作用等特點，在發(fā)酵肉制品中應用很多。,同時，也廣泛用于發(fā)酵植物性食品,.,曲冬梅,劉小杰,植物乳桿菌及其在食

45、品工業(yè)中的應用,?,J Nutr. 2011 Apr 13. Epub ahead of print,?,Lactobacillus johnsonii,N6.2 Stimulates the Innate,Immune Response through Toll-Like Receptor 9 in,Caco-2 Cells and Increases Intestinal Crypt Paneth,Cell Number in BioBreeding Diabetes-Prone Rats.,?,Lactobacillus johnsonii,(Ljo) N6.2 has been sho

46、wn to,mitigate,（減輕）,the development of type 1 diabetes,when administered to diabetes-prone rats. The specific,mechanisms underlying this observed response remain,under investigation. The objective of this study was to,assess the effect of Ljo N6.2 on mucosal,（粘膜的）,inflammatory response using differe

47、ntiated Caco-2,monolayers. The mRNA expression levels of CCL20,CXCL8, and CXCL10 chemokines,（趨化因子）,were,determined by qRT-PCR. Ljo at 10(11) CFU/L induced a,strong response in all chemokines examined.,The Caco-2 monolayer is widely used across the pharmaceutical industry as an in vitro model of the

48、human,small intestinal mucosa,粘膜,to predict the absorption of orally administered drugs,?,To assess the specific host-signaling pathways involved,we performed RT-PCR amplification of Toll-like,receptors (TLR) and nucleotide-binding oligomerization,domain-like receptors. TLR7 and TLR9 expression leve

49、ls,were induced 4.2-,and 9-fold, respectively, whereas,other TLR and nucleotide-binding oligomerization,domain receptors were not modified. A similar effect was,observed in Caco-2,monolayers treated with Ljo cell-free,extract or purified nucleic acids (NA).,Increased levels of,IFN type 1 and IFN reg

50、ulators Stat1 and IRF7 followed,the upregulation of TLR9. Activation of TLR9 was also,evidenced by increased Frizzled 5 expression in Ljo-,treated Caco-2 cells and an increase in the number of,Paneth cells in Ljo-fed, diabetes-prone rats.,Paneth,細胞是一種分布于腸腺底部的腸黏膜分化上皮細胞,可向腺體分泌生長因子和抗微生物肽,如溶菌酶和防御素,.,隨著對

51、炎癥性腸病,(IBD),發(fā)病機制研究的不斷深入,已發(fā)現(xiàn),Paneth,細胞及其分泌,的抗微生物分子參與了腸道黏膜屏障的破壞,可致自身免疫失衡,啟動腸道炎癥反應,從而引起腸道,黏膜功能的改變,在,IBD,的觸發(fā)和復發(fā)中起一定作用,.Paneth,細胞在,IBD,中是參與免疫防御還是促進,免疫損傷,目前仍在研究討論中,.,?,These results are in agreement,with the,polarizing-tolerizing mechanism recently,described in which the apical,（頂?shù)模?stimulation of TLR9 in

52、 intestinal epithelial,cells leads to a higher state of,immunologic alertness. Furthermore,these results suggest that live probiotics,could be, in the future, replaced with select,cellular components.,Yogurt and longevity,?,加利亞有三寶：玫瑰花、葡萄酒，還有酸奶。酸奶不僅給,保加利亞人帶來了美味，還帶來了健康。保加利亞人的長,壽老人比例在同地區(qū)中最高，各種疾病的發(fā)病率也相對

53、較,低。早在上個世紀初，俄國著名科學家米奇尼科夫在研究,了世界,30,多個國家人口壽命情況后發(fā)現(xiàn)，保加利亞的百歲,老人比例最高。他發(fā)現(xiàn)保加利亞人的膳食結構里，飲用酸,奶的數(shù)量居各國之首。保加利亞的一名醫(yī)學家格里戈羅夫,聞悉后，經(jīng)過多次試驗，發(fā)現(xiàn)保加利亞酸奶中含有一種特,殊的益生菌，這種菌在酸化鮮奶的同時使奶稠化而不變質，,促進腸胃中食物殘渣的分解和吸收，還能分解乳蛋白，抑,制腸胃中致病菌的生長。這種菌后來被命名為“保加利亞,乳酸桿菌”。而現(xiàn)在的高品質酸奶里幾乎都少不了保加利,亞乳酸桿菌的身影。,2006,年，為紀念格里戈羅夫的發(fā)現(xiàn)，,人們在其故鄉(xiāng)特倫市建起了酸奶博物館。,保加利亞酸奶皇后,La

54、ctobacillus,bulgaricus,（保加利亞乳桿菌）,?,Anaerobic, growth temperature 37-45,.,?,Dairy products, including yogurt,cheese, etc.,Genomic studies of,Lactobacillus,bulgaricus,?,Proc Natl Acad Sci USA. 2006, 103(24):,9274,9279.,?,The complete genome sequence of,Lactobacillus bulgaricus,reveals,extensive and on

55、going reductive,evolution.,?,Lactobacillus delbrueckii,ssp.,bulgaricus,(,L.,bulgaricus,) is a representative of the group of,lactic acid-producing bacteria, mainly known for,its worldwide application in yogurt production.,The genome sequence of this bacterium has,been determined and shows the signs

56、of,ongoing specialization, with,a substantial,number of pseudogenes and incomplete,metabolic pathways and relatively few regulatory,functions,. Several unique features of the,L.,bulgaricus,genome support the hypothesis that,the genome is in a phase of rapid evolution.,?,(,i,) Exceptionally high numb

57、ers of rRNA,and tRNA genes,（,95,）,with regard to,genome size may indicate that the,L.,bulgaricus,genome has known a recent,phase of important size reduction, in,agreement with the observed high,frequency of gene inactivation and,elimination;,(,ii,) a much higher GC content,at codon position 3 than e

58、xpected on the,basis of the overall GC content suggests,that the composition of the genome is,evolving toward a higher GC content;,?,and (,iii,) the presence of a 47.5-kbp inverted,repeat (,反向重復序列,) in the replication,termination region, an extremely rare feature in,bacterial genomes, may be interpr

59、eted as a,transient stage in genome evolution.,The results,indicate the adaptation of,L. bulgaricus,from a,plant-associated habitat to the stable protein and,lactose-rich milk environment through the loss of,superfluous (,多余的,) functions and,protocooperation (,互生,) with,Streptococcus,thermophilus,(,

60、嗜熱鏈球菌,) .,Lactobacillus acidophilus,(,嗜酸乳桿菌）,?,Lactobacillus acidophilus,is probably the best,well-known species of,Lactobacillus, it is,naturally found in the human and animal,gastrointestinal (GI) tract (,胃腸管道,), mouth and,vagina,（陰道）,.,?,It has been suggested that,L. acidophilus,is a,beneficial o