Antibiotic Biosynthesis Following Horizontal Gene Transfer .
PerspectiveAntibiotic biosynthesis followinghorizontal gene transfer: newmilestone for novel naturalproduct discovery?1.Introduction2.Rhodostreptomycin, a novelantibiotic biosynthesisedfollowing horizontal genetransfer from Streptomyces toRhodococcus3.Expert opinionKazuhiko Kurosawa, Daniel P MacEachran & Anthony J Sinskey†Massachusetts Institute of Technology, Department of Biology, and Engineering Systems Divisionand Health Sciences & Technology, Cambridge, MA, USABacteria obtain a significant proportion of their genetic diversity via acquisition of DNA from distantly related organisms, a phenomenon known as horizontal gene transfer. The focus of horizontal gene transfer investigationshas been primarily on the impact of this phenomenon on the ecologicaland/or pathogenic characteristics of bacterial species, with very little effortdevoted to investigating horizontal gene transfer as a means of drug discovery. Here, we describe a novel approach to harness the power of horizontalgene transfer to produce novel chemotherapeutic molecules, a process that,is easily scalable. We describe the state of the art in this field andad discussolthe current limiting factors associated with this phenomenon.Utilising awn e.horizontal gene transfer method, we have identified anddocharacterisedasluan atermednovel antimicrobial compound. Production of this antibiotic,rhodoscnser erasospecies of Streptotreptomycin, is associated with the transfer of DNAsfromud mechanism.rpmyces to Rhodococcus by an as yet identifiedWe believeise y forothat horizontal gene transfer may representthefutureofnaturalproductpt h couediscovery and engineering.A ldLt ionKutUbaritmisorfDlInai01rc0e2m mto d ghCgr hibite t a sinri drug discovery,oyKeywords: antibiotic,rohorizontalrin gene transfer, natural productpepploe nda (2010)us 5(9):819-825aExpertCOpin. Drug Discov.Sde iewrsivfo uthor lay,1. Introductionto Una dispNIt has become increasingly evident that horizontal gene transfer plays a crucial rolein microbial adaptation, speciation and evolution, as well as in contributing to amyriad of environmental and public-health problems [1,2]. Horizontal gene transferenables bacteria to reorganise their genomes and to quickly acquire novel featuressuch as pathogenicity factors, drug resistance and metabolic properties [3-5]. Horizontal gene transfer between prokaryotes is relatively well understood and occursvia three fundamentally distinct mechanisms [5,6]: transformation, in which a celltakes up foreign DNA molecules from the surrounding medium; conjugation,which involves the direct transfer of DNA from one cell to another; and transduction, in which DNA transfer is mediated by bacterial viruses. These procedures havebecome standard molecular biology tools. However, a mechanism by which a natural horizontal gene transfer process induces antibiotic production has not beendocumented, although there is indirect evidence that such processes occur [7-9].The focus of the majority of horizontal gene transfer investigations has been primarily on the impact of this phenomenon on the ecological and/or pathogenic characteristics of bacterial species. Very little effort has been devoted to investigatinghorizontal gene transfer as a means of drug discovery.The key to developing effective therapeutics is the identification of novelcompounds and chemical structures that increase the chemical diversity available10.1517/17460441.2010.505599 2010 Informa UK, Ltd. ISSN 1746-0441All rights reserved: reproduction in whole or in part not permitted819
Antibiotic biosynthesis following horizontal gene transferfor study at the molecular level for targeted therapies. Secondary metabolites produced by microorganisms and plants offermolecular diversity with the opportunity to create new molecules that form the basis of important drugs. However, it isgenerally assumed that the majority of these secondary metabolite compounds have already been discovered and that we arelimited to the conventional techniques in use to search fornew natural products. Thus, we postulate that a paradigmswitch is necessary to identify novel secondary metabolitesthat have therapeutic value. Identifying effective strategies todiscover and exploit new bioactive molecules is integral tothe success of future drug discovery efforts.Recently, we isolated and characterised a strain of Rhodococcus that previously was not able to produce antibiotics but hadacquired the capacity to biosynthesise a novel antibiotic following competitive co-culture between Rhodococcus and astrain of Streptomyces [10]. In this article, we highlight recentprogress and discuss future prospects for the possible exploitations of new methodologies for the discovery of novelchemical entities.2. Rhodostreptomycin, a novel antibioticbiosynthesised following horizontal genetransfer from Streptomyces to RhodococcusCo-cultivation of Rhodococcus and StreptomycesStreptomyces are well known as producers of antibiotics [11,12].The actinomycete Rhodococcus, a relative of Streptomyces, hasonly rarely been reported to produce antibiotics [13]. Interestingly, multiple polyketide synthase genes have been identified in the various Rhodococcus genomes suggesting thatthese organisms possess the capability to produce antibioticcompounds [14]. In Streptomyces, it has been shown that differentiation and induction of secondary metabolism occurswhen the cells encounter adverse environmental conditions [15,16]. Previous reports have demonstrated that someribosomal mutations, specifically mutations in the rpsLgene encoding ribosomal protein S12, result in a dramaticactivation of antibiotic production. Thus, one can readilymodulate ribosome function, and subsequently antibioticproduction, by selecting for mutations within the ribosomalproteins using antibiotics that specifically target thesestructures [17]. To address the possibility that stressinduced mutations might activate a latent antibiotic biosynthesis pathway in Rhodococcus, physiological stresses andexternal biological stimuli were imposed on Rhodococcus fascians DDO356, a strain in which we have not previouslydetected antibiotic production. In order to achieve thisgoal, spontaneous R. fascians DDO356 streptomycinresistant (str) mutants were selected. Forty-two of the strmutants were arbitrarily chosen and screened for antibioticproduction. Cultures of these strains did not display antimicrobial activities. In additional experiments, cells of strain121, the DDO356 str-strain that revealed the highest levelof streptomycin-resistance (10 mg ml-1), were spread onto2.1820solidified peptone--yeast extract-iron salts medium (ISP 2medium) supplemented with rifampicin. In all, 62 str-rifdouble mutants were isolated, yet none of these strains produced detectable antibiotics. As an additional environmentalstress, strain 307, one of the double mutants which displayedresistance to as much as 0.3 mg ml-1 rifampicin, was cocultured competitively with the actinomycin-producingStreptomyces padanus MITKK-103 [18]. The cultures werecombined in baffled flasks with various ratios of strain307 and MITKK-103, and then cultured on GS mediumconsisting of 1% soluble starch, 2% glucose, 2.5% soytone,0.4% dry yeast, 0.1% beef extract, 0.005% K2HPO4 and0.2% NaCl, pH 7.0, on a rotary shaker at 30oC. After5 days of co-culture, mycelia from each of the flasks werestreaked onto ISP medium 2 plates and incubated at 30oCfor an additional 7 days to elucidate the behaviour of theculture. Colonies appearing on the plates were predominantly Rhodococcus, but a few colonies of Streptomyces arosefrom every co-culture except for one (Figure 1A 3). The oneplate, streaked from a flask culture that had been inoculatedwith a 5:15 ml ratio of Rhodococcus and Streptomyces, produced only Rhodococcus cells, and those cells exhibited achange in colony morphology (Figure 1A 4). Astonishingly,all of the Streptomyces had been eliminated from the culture.Ten Rhodococcus colonies from the plate were randomlychosen, cultured on GS medium at 30oC for 5 days andscreened for antibiotic production. Interestingly, one of theisolates, designated strain 307CO, generated antimicrobial activity against Escherichia coli, Staphylococcus aureus,Bacillus subtilis and S. padanus [10].Product analysis of Rhodococcus variantsTo evaluate the differences between the DDO356 variants,strain 121, strain 307, and strain 307CO were each culturedand characterised by HPLC. Among these, only strain307CO produced an antibiotic with especially potent antimicrobial activity against S. padanus. As the bioactive fractionwas detected in the supernatant, 10 ml of the supernatantfrom each culture was extracted twice with an equal volumeof n-butanol. The butanol was removed and the residualmaterial was dissolved in 1 ml of 5% acetonitrile in waterwhich was then subjected to reverse-phase HPLC. Theseresults demonstrated differences in the product profiles ofthe DDO356 variants (Figure 1B 1--5). The retention timeof detected peaks from wild-type DDO356 (Figure 1B 2),strain 121 (Figure 1B 3) and strain 307 (Figure 1B 4) were similar, although the areas under some of the peaks from strain121 and strain 307 were higher than those from DDO356,suggesting that these strains had more active secondary metabolism in association with their acquired resistance to streptomycin and rifampicin. On the other hand, several peaks thatwere not detected in the DDO356, 121 or 307 extractsappeared at the retention time of 3 -- 7 min and15 -- 17 min in 307CO (Figure 1B 5). A bioactive componentwith antimicrobial activities was eluted at the retention time2.2Expert Opin. Drug Discov. (2010) 5(9)
Kurosawa, MacEachran & NHONHHNHONH2HOOHHNNHNH2Rhodostreptomycin e (220 30.20.10OHHNNHNH250Rhodostreptomycin B1020304050Time (min)Figure 1. Product analyses of Rhodococcus fascians ariants. A. Colonies arising from competitive co-culture of Rhodococcusand Streptomyces. 1, Streptomyces padanus MITKK-103 alone; 2, R. fascians strain 307 (str-rif-resistant mutant of R. fasciansDDO356) alone; 3 and 4, co-cultured flask of R. fascians strain 307 and S. padanus MITKK-103. While we generally observedboth Rhodococcus and Streptomyces in the co-culture experiments (3), in one co-culture flask we were only able to isolateRhodococcus. B. HPLC profiles of the extracts. 1, medium alone control; 2, wild-type DDO356; 3, strain 121, str mutant ofDDO356; 4, strain 307, rif mutant of strain 121; 5, strain 307CO, isolated from a co-culture of strain 307 and S. padanusMITKK-103. Product analysis was performed by an analytical HPLC (Hitachi D-7000 system) using a J.T. Baker Octadecyl (C18)SP-120 column (5 µm, 4.6 250 mm); mobile phase, 5% CH3CN in 0.05% H3PO4 at 0 -- 20 min and 5 -- 100% CH3CN gradient in0.05% H3PO4 at 20 -- 50 min; flow rate, 1 ml/min. C. Structures of rhodostreptomycins, antibiotics produced by strain 307CO.of 3 -- 5 min. Using assay-guided fractionation, the bioactivecompounds were purified by a combination of cationexchange and reversed-phase HPLC. Ten litres worth of theculture broth had to be combined to purify and identify thecompounds. Two compounds were characterised, assigned amolecular formula of C22H40N8O13, and shown to belongto a new group of aminoglycosides (Figure 1C), which wehave since named rhodostreptomycin [10].Genome analysis of Rhodococcus variantsThe product profile in strain 307CO suggested that a genomic alteration might have occurred in Rhodococcus as a resultof the competitive co-culture with Streptomyces. Pulsedfield gel electrophoresis (PFGE) analysis of the genome ofwild-type Rhodococcus DDO356 and the isolated variants supported this hypothesis. Agarose-embedded DNA from thevariants cultured under identical conditions for 48 h was2.3Expert Opin. Drug Discov. (2010) 5(9)821
Antibiotic biosynthesis following horizontal gene transferM123456A.71234567B.(kb)436.5 –194 –48.5 –Figure 2. PFGE and Southern blot analyses of Rhodococcus variants and Streptomyces padanus MITKK-103. A. PFGE.B. Southern hybridisation. Lanes: 1, DNA from 48 h culture of Rhodococcus fascians DDO356; 2, DNA from 48 h culture ofstrain 121; 3, DNA from 48 h culture of strain 307; 4, DNA from 48 h culture of strain 307CO; 5, DNA from 16 h culture ofStreptomyces; 6, DNA from 24 h culture of Streptomyces; 7, DNA from 96 h culture of Streptomyces. M indicates l ladder asmolecular mass markers. Hybridisation was carried out using a digoxigenin-labelled fragment (230-bp) derived from theStreptomyces megaplasmid isolated from the 24 h culture.PFGE: Pulsed-field gel electrophoresis.electrophoresed under conditions that allow separation oflinear fragments between 30 and 600 kb. A large extrachromosomal element (megaplasmid) of around 50 kb that wasabsent in the wild-type DDO356, 121 and 307 strains wasfound in 307CO (Figure 2A, lane 1--4). DNA from S. padanusMITKK-103 that had been cultured for 16, 24 or 96 hrevealed one or more megaplasmids ranging in mobilityfrom 160 to 380 kb (Figure 2A, lane 5--7). We postulatedthat the observed shift may be the result of intercalationof actinomycins, which are synthesised and secreted byS. padanus, within the plasmid DNA. To address this possibility, we incubated the purified megaplasmid in S. padanuscultured media. We found that we could reliably reproducethe mobility shift under these conditions suggesting thatthe observed shift was in fact due to a compound secretedby the bacterium. The shift in the apparent molecularmass of the megaplasmid from S. padanus MITKK-103 ismost likely due to the accumulation of actinomycins in thisstrain. To elucidate the relationship between the Rhodococcusmegaplasmid and the Streptomyces-megaplasmid, Southernhybridisation was carried out using a probe containing a fragment derived from the Streptomyces megaplasmid (which wecall pSPM1) that had been cultivated for 24 h. Southernhybridisation was carried out using the DIG high primeDNA labelling and detection starter kit II (Roche). ProbeDNA was labelled with digoxigenin-dUTP and hybridisationconditions, washing and detection were performed accordingto the manufacturer’s instruction. Megaplasmid DNA was822purified from a pulsed-field gel using b-Agarase I (New England Bio Labs) and digested by PspOMI. Restriction fragments were ligated into the NotI site of pCR2.1-TOPO andtransformed into competent E. coli cells, as per the manufacturer’s instructions (Invitrogen). After selecting and analyzingcolonies, the plasmids were digested by ApaI and restrictionfragments isolated from several clones were prepared as probesfor Southern hybridisations. When one (a fragment of230-bp: the nucleotide sequence of which has been depositedin GenBank under the accession number EU247609) of thefragments was used as a probe, hybridisation of the labelledDNA fragment against the filter with the 307CO megaplasmid (50 kb) revealed a signal, as shown in Figure 2B, lane 4.This indicated that the megaplasmid of 307CO possesseshomology to a portion of pSPM1. We were unable to detecthybridisation using any of the other Rhodococcus variants(Figure 2B, lane 1--3). Subsequent analysis of DNA fragmentsderived from the 307CO megaplasmid showed them to beidentical to fragments derived from Streptomyces, indicatingthat 307CO had obtained pSPM1 DNA from Streptomyces.To determine the identity of the DNA transferred fromStreptomyces to Rhodococcus, a cosmid library of the pSPM1megaplasmid was constructed in the cosmid vector SuperCos1(Stratagene), and screening was carried out using the aforementioned probe (the fragment of 230-bp) as we knew thisregion was positively correlated to the aforementioned antibiotic production. A positive cosmid clone (cosmid 24) wasisolated and sequenced. The cosmid bore a 24416-bp insertExpert Opin. Drug Discov. (2010) 5(9)
Kurosawa, MacEachran & SinskeyA.Original 230-bp fragment048121624 kbR20LPCR (I)4656PCR (III)8692 1064013944PCR (II)7559B.5 kbp3 p1 kbp-Figure 3. Detection of the Streptomyces-derived DNA in Rhodococcus strain 307CO. A. Diagram of the 24416-bp fragmentcloned from the Streptomyces padanus MITKK-103 megaplasmid pSPM1 into cosmid 24. The probe described in Figure 2 islabelled as ‘original 230-bp fragment’. B. Amplification products obtained by PCR of genomic DNA from strain 307, strain307CO and S. padanus MITKK-103 using the primer pairs 4656f/8692r (I), 7559f/10610r (II) and 10640f/13944r (III) shown inFigure 3A. Lane 1, strain 307; lane 2, strain 307CO; lane 3, S. padanus MITKK-103. M indicates l ladder as molecularmass markers.with a GC content of 68.3%, revealing a total of at leastnine open reading frames (ORFs) (Figure 3A). BLAST analysisof protein databases (blastx) using the obtained sequencerevealed that six of the ORFs have homologies with knowngenes: a putative acetyltransferase from Streptomyces hygroscopicus(GenBank accession no. ZP05517589.1), a transcriptionalregulator from Streptomyces flavogriseus (ZP05806390.1), aresolvase domain protein from Nakamurella multipartita(YP003202886.1), a putative transcriptional regulator fromStreptomyces viridochromogenes (ZP05529456.1), a threoninedehydratase from Streptomyces albus (ZP04703027.1) anda DNA integrase/recombinase from Rhodococcus jostii(YP708625.1) with 62, 45, 68, 73, 62 and 57% identities,respectively. The remaining ORFs are homologous to hypothetical proteins from Streptomyces sp. (YP001661505.1), Streptomyces clavuligerus (ZP05008406.1) and Streptomyces sp.(ZP06272626.1) with 42, 43 and 69% identities, respectively.To confirm the presence of the Streptomyces-derived DNA inRhodococcus strain 307CO, PCR was conducted on both thevarious Rhodococcus strains. While no PCR products wereobtained from Rhodococcus strain 307 genomic DNA, thePCR products amplified from genomic DNA of theStreptomyces and Rhodococcus strain 307CO were of theexpected size (4037-, 3052- and 3305-bp) (Figure 3B, lane1--3). The PCR products were each sequenced, and it wasfound that all the sequences obtained from the two strains sharean identity of 99.9% (data not shown). These data suggestthat Rhodococcus had taken up DNA from the Streptomyces,including at least a 9.2 kbp fragment of a Streptomyces megaplasmid. In subsequent PFGE experiments, mobility of themegaplasmid from strain 307CO varied between culturebatches despite growth under identical conditions. The erraticbehaviour of the Rhodococcus megaplasmid observed usingPFGE suggested that this genetic element is unstable in thenew host, thus, resulting in instability of the antibioticproducing phenotype. Loss of the Streptomyces-derived DNAcorrelated with loss of the ability of Rhodococcus strain307CO to produce rhodostreptomycins, showing a correlation between antibiotic production and presence of theStreptomyces DNA in Rhodococcus. In some cases, strain307CO also appeared to lose the capacity to produce theantibiotic on multiple rounds of subculturing (data notshown). It is noteworthy that the antibiotics biosynthesisedin Rhodococcus following horizontal gene transfer fromExpert Opin. Drug Discov. (2010) 5(9)823
Antibiotic biosynthesis following horizontal gene transferStreptomyces are aminoglycosides and differ widely in thestructure from actinomycins, polypeptide antibiotics that areproduced by Streptomyces [10]. How those phenomena influence antibiotic production in Rhodococcus strain 307COremains unclear and will require further experimentation.While the underlying molecular and biochemical mechanisms responsible for rhodostreptomycin biosynthesis remainelusive, there is no doubt that the transfer of DNA fromStreptomyces to Rhodococcus is associated with the observedbiosynthesis of rhodostreptomycin. The route of this transfer,transduction, conjugation or transformation is unknown butone can readily ru
1. Introduction 2. Rhodostreptomycin, a novel antibiotic biosynthesised following horizontal gene transfer from Streptomyces to Rhodococcus 3. Expert opinion Perspective Antibiotic biosynthesis following horizontal gene transfer: new milestone for novel natural product discovery? Kazuhiko Kurosawa, Daniel P MacEachran & Anthony J Sinskey†
and the Core Elements of Antibiotic Stewardship for Nursing Homes (23). This 2016 report, Core Elements of Outpatient Antibiotic Stewardship, provides guidance for antibiotic stewardship in outpatient settings and is applicable to any entity interested in improving outpatient antibiotic prescribing and use.
R M AB ARP R R M APP B 3 Completeness of antibiotic prescribing documentation. Ongoing audits of antibiotic prescriptions for completeness of documentation, regardless of whether the antibiotic was initiated in the nursing home or at a transferring facility, should verify that the antibiotic prescribing
the biosynthesis of chlorophylls (phytyl side-chain) and carotenoids, and differentiates it from the cytosolic ac-etate/mevalonate pathway of sterol biosynthesis. The light-induced accumulation and final functional con-centration of carotenoids and chlorophylls in green leaves is presented. Moreover, the biosynthesis of isoprene and
Isoprenoids Fig. 1 The relationship between alginate biosynthesis and the cellular metabolism in P. fluorescens. a The proteins and metabolites needed for alginate biosynthesis. b A simplified model of the cell's metabolism highlighting the processes identified in the present study as being important for full alginate biosynthesis levels.
One Gene-One Enzyme Hypothesis (Beadle & Tatum) The function of a gene is to dictate the production of a specific enzyme One Gene—One Enzyme but not all proteins are enzymes those proteins are coded by genes too One Gene—One Protein but many proteins are composed of several polypeptides, each of which has its own gene One Gene—One Polypeptide
biosynthesis of steroidal alkaloids in Solanaceae plants. We found that GAME9 is part of an ERF-gene cluster existing in potato and tomato. Transactivation and promoter binding assays as well as transgenic tomato and potato plants revealed that GAME9 controls SGA biosynthesis as well as several upstream mevalonate and cholesterol precursor .
to govern lantibiotic biosynthesis (Scheme 2). The key difference expected between the cle and the med clus-ters was the presence of an essential amidinohydrolase gene, encoding an enzyme that would act at a late stage in the path-way to unmask the primary amino group of the mediomycins, as we have previously described for the biosynthesis of .
Anaesthetic Machine Anatomy O 2 flow-meter N 2 O flow-meter Link 22. Clinical Skills: 27 28 Vaporisers: This is situated on the back bar of the anaesthetic machine downstream of the flowmeter It contains the volatile liquid anaesthetic agent (e.g. isoflurane, sevoflurane). Gas is passed from the flowmeter through the vaporiser. The gas picks up vapour from the vaporiser to deliver to the .
