Study On The Replication Mechanism Of The Mega-plasmid Of Streptomyces Cattleya And Type ? Toxin-antitoxin Systems

Streptomyces cattleya is known as the model bacterium to study the biosynthesis of thienamycin and fluoro-metabolites.By pulsed-field gel electrophoresis(PFGE),two replicons with size over 1.6 Mb were observed in the genome of S.cattleya DSM46488,which is rare in other Streptomyces species.In this study,we focused on the special genome structure and replicative mechanism of the smaller replicon was determined by genomics,bioinformatics,and molecular genetics approaches to explore the evolution relationship of the two giant linear replicons.Firstly,the special genome structure of S.cattleya DSM46488 was confirmed by genome sequencing and analysis(Chapter 3).The whole genome sequence of S.cattleya DSM46488 was obtained by 454 pyro-sequencing.After the assembly,two large linear replicons were obtained.The larger one showed a size of 6.28 Mb,and the other one was 1.81 Mb,which was consistent with the result of the previous PFGE results.The smaller replicon was proposed to be a mega-plasmid with the evidences from genome analysis,including:(i)There was no rRNA,tRNA or other primary metabolism-related proteins encoded in the 1.81 Mb replicon;(ii)Comparing with the core regions of other sequenced Streptomyces,no conserved gene was identified in the 1.81 Mb replicon;(iii)Phylogenetic analysis result based on the partition proteins showed that both of ParA and ParB proteins(SCATT_p08020 and SCATT_p08030)encoded by the 1.81 Mb replicon were clustered with those encoded by other Streptomyces plasmids,while its chromosomal ParA protein(SCATT 31180)was clustered with other Streptomyces chromosomal Par proteins.On the other hand,there is cross-interaction between the megaplasmid and chromosome.For example,the essential genes responsible for the biosynthesis of fluoro-metabolites scattered within the two linear replicons,and the two replicons shared a novel telomere system Additionally,an actinomycete integrative and conjugative element(AICE)ICEScaDSM46488-1 was identified in the linear chromosome.When incubated in TSBY,YEME or MM medium,ICEScaDSM46488-1 could be excised from the chromosome without induction.However,different medium and growth phases exhibit a significant effect on the excision frequencySecondly,the replication mechanism of the mega-plasmid pSCATT was explored by studying the telomere system(Chapter 4).As the secondary structure in telomeres would result in the incomplete terminal sequence obtained by 454 pyro-sequencing,the probable telomeres of S.cattleya DSM46488 were cloned and sequenced individually.Non-archetypal telomeres were obtained,including the two telomeres cpTelo and pTelo of the megaplasmid and cpTelo and cTelo of the chromosome,of which the cpTelo was shared by megaplasmid and chromosome.Although there was no homology between the S.cattleya telomeres and archetypal telomeres on the level of both sequence or secondary structure,all of the telomeres contained a promoter functional in E.coli,which was consistent with the formerly reported archetypal telomeres.Thus,we presumed that S.cattleya DSM46488 harbored a novel telomere system that was rarely presented.In pSCATT,there were two novel pairs of terminal proteins and telomere-associated proteins encoded by tapl-tpgl(SCATT p17400-SCATT_p17410)and tap2-tpg2(SCATT_p03430-SCATT_p03440-SCATT_p03450)The genetics evidence revealed that tapl-tpgl and tap2-tpg2 were responsible for the replication of telomeres pTelo and cpTelo,respectively.Different to the known telomeric protein systems,tpg2 locates upstream of tap1.SCATT p03430 of tap2-tpg2 was shown to be unnecessary,and further work is needed to determine the function of SCATT p03430In addition,one replication origin of the mega-plasmid pSCATT was predicted and characterized(Chapter 4).The replication origin was about 2 kb,which was in the centre of pSCATT and upstream of the ParAB operon.The recombinant plasmid containing a 2 kb fragment was determined to be replicative in S.lividans.One ORF(SCATT p08010)and the upstream non-coding sequence were included in this DNA fragment.Although the replication origin of pSCATT shows no sequence similarity to the other known replication origin,there are common features of replication origin:(I)The replication initiator protein SCATT_p08010 contained an HTH domain,and the EMSA results showed that SCATT-p08010 was capable of interacting with the upstream non-coding sequence.(?)In the non-coding sequence,two pairs of 13-bp direct repeats and two pairs of 10-bp inverted repeats were identified.However,the identified replication origin endows pSCATT only the capacity of circular replication but not linear in Streptomyces.It is speculated that other supplementary genes might be needed when the identified replication origin was employed to replicate in linear mode,or there might be other replication origins responsible for the linear replication of pSCATT.Finally,research on type ? toxin-antitoxin system(TA system)of S.cattleya DSM46488 was also involved in the present study(Chapter 5).Type II toxin-antitoxin system,which is usually encoded by an operon made up of genes for toxin and antitoxin proteins,is widely spread in bacteria.TA systems have been reported to be contributive to the genetic stability of plasmids,persistence cells formation and other physiological processes.However,there are few reports on this topic in Streptormyces.33 putative type ? TA systems are identified in the S.cattleya DSM46488 genome,including relBE,vapBC,and phd-doc families.When the toxin genes were expressed in E.coli,RelBE family TA locus relBE2sca(SCATT 39270-SCATT 39280)was found functional.Under unfavorable conditions,such as high osmotic pressure,the antitoxin RelB2sca would be degraded by S.cattleya encoded proteinase complex ClpPX,causing the release of the toxin RelE2sca from the RelBE2sca complex and finally activating the relBE2sca system.It was worth noting that E.coli encoded RelBeco can alleviate the toxicity of RelE2sca,indicating that there is cross-interaction among the non-cognate relBE systems even encoded by different species.However,the wild type RelB2sca can't alleviate the toxicity of RelEeco unless the Asn61 and Met68 are replaced by Val and Leu,respectively.There is no protein homologous to RelBE2sca identified in S.lividans TK24,S.avermitilis MA4680,and S.sp.FR008.The fact that relBE2sca was capable of working as a bona fide TA locus in the commonly used Streptomyces implied that it has the potential to be developed into a regular genetic selection marker.In conclusion,the genome structure of S.cattleya DSM46488 containing two giant linear replicons was confirmed in this study.Two novel pairs of telomere systems and one replication origin were identified in S.cattleya mega-plasmid pSCATT.Additionally,the first Streptomyces encoded RelBE family TA system was identified as well.The work will be potentially helpful to study the evolutionary relationship of S.cattleya to other important Streptomyces genomes.