This week’s BEACON Researchers at Work blog post is by University of Idaho postdoc Wesley Loftie-Eaton.
I stumbled into the world of plasmids at my alma mater, the University of Stellenbosch in South Africa. My advisor, Prof. D. E. Rawlings, asked me to determine the biological implications of a very specific mutation within the origin of replication of two isogenic plasmids (Loftie-Eaton and Rawlings, 2009; Loftie-Eaton and Rawlings 2010). What started as a very simple and specific question quickly enthralled me with its true complexity. During that time I learned that you can’t understand plasmids, or anything in biology for that matter, without the context of evolution; and what better place to study plasmid evolution than at the University of Idaho alongside Dr. Eva M. Top and under the umbrellas of IBEST and BEACON.
In case you’re not a plasmid geek like me, plasmids are generally circular (linear forms also exist) DNA entities that replicate autonomously in bacteria. The smallest plasmids are but a few hundred base pairs while the largest are over a million base pairs long (Kado et al., 1998). Based on sequence information, about half of plasmids are either self-transferable by means of conjugation or can be transferred (mobilized) by conjugative plasmids, while the remainder appear to be non-transferable (Smillie et al., 2010). Many plasmids are cryptic or do not encode any non-essential genes while others may carry genes that provide benefit to the bacterial host, allowing it to occupy and proliferate in an otherwise hostile niche (Kado et al., 1998). Depending on whether a plasmid provides its host with benefit or cost, they can be likened to symbionts or parasites, respectively, and like symbionts and parasites, they have a host range that is either narrow or broad.
Projects I am currently working on are focused on a) elucidating the molecular mechanisms that evolve to permit plasmids to shift, contract or expand their host range and b) to understand how broad host range plasmids proliferate and spread in complex microbial communities. The latter will not be discussed here. Besides the fundamental nature of these questions, they are relevant to human health due to the rampant plasmid-mediated spread of antibiotic resistance amongst pathogens.
When a plasmid conjugates to a naïve host it may not necessarily persist unless there is selection for its maintenance in that host. Failure to persist could be due to inefficient replication, poor partitioning or segregational loss of the plasmid during cell division, or plasmid-containing cells can simply be outcompeted by plasmid-free cells if the plasmid imposes a cost on the host. We know, however, that plasmid and host can rapidly adapt to each other while under strong selection for plasmid maintenance, after which the plasmid can continue to persist for prolonged periods even when the strong selection is removed (Bouma and Lenski, 1988; Dahlberg and Chao, 2003; Sota et al., 2010). Panels A and B in the figure below demonstrate exactly this; an initially unstable plasmid-host relationship evolved a more stable phenotype in less than 200 generations and after ~600 generations the plasmid was highly stable in that host1. Not clear from this figure (due to me not showing the full assay data) was that in the absence of antibiotic selection the ancestral plasmid tended towards extinction, however, a persistent relationship in which the plasmid was maintained in ~10% of the population due to horizontal transfer evolved within the first 100 generations (panel C, below)! Together with collaborators Sam Hunter and Haruo Suzuki I am currently working on elucidating the genetic changes behind this increase in persistence and collaborator Jose Ponciano is working on means to quantify how easy or difficult it is to switch between trajectories of persistence and extinction.
In another experiment (different plasmid and host)2 we found that a transposon encoded on a plasmid native to that host ‘jumped’ to the introduced plasmid, which was being maintained under antibiotic selection. The result was increased stability of the introduced plasmid, even in the absence of antibiotic selection, and loss of the native plasmid. Encoded on the transposon are a toxin-antitoxin (TA) system and a resolvase, both of which we have now shown to promote plasmid stability (Loftie-Eaton et al., in prep.). Even more significant, however, was that the evolved plasmid that acquired the transposon was completely stable in other beta- and gamma-proteobacteria in which its ancestor was unstable. Thus, acquisition of a transposon encoding stability functions resulted in an apparent expansion of the plasmid’s host range and more broadly, we demonstrated the interplay and fate of genes that could arguably be labeled as “selfish”.
In yet another system one of the outcomes was a deletion mutation within the plasmid’s origin of replication. Though much work remains to be done, this mutation has me extremely excited. The type and location of the mutation is the same as the mutation I set out to understand during my PhD. The exciting part is that the plasmids I studied then belong to a different family and evolved in the environment, whereas here the mutations occurred during experimental evolution in the lab. Preliminary results showed that this deletion abolished the plasmid’s ability to replicate in a previously permissive host, which already is novel information, however, if my hypothesis based on my previous research withhold scrutiny, then these results will demonstrate that what we observe during experimental evolution in the lab also occurs in nature, and vice versa. Validation!
Though far from complete, what we’ve learned thus far is that plasmid host-range can evolve quite rapidly, that such rapid changes tend to occur through mutations that result in gain or loss of function and that there are multiple molecular solutions that can lead to stable plasmid-host relationshi
ps. From the perspective of a plasmid geek this is fascinating, but from a medical perspective it’s concerning; once a multidrug resistance plasmid has established in a population it intends to stay, even if the antibiotics that initially selected for its maintenance are removed from the system! However, by accumulating more data of this kind we hope to define general patterns in the evolution of plasmid host range that can aid in the development of novel drugs to inhibit the spread and establishment of multidrug resistance plasmids.
1The experimental work was done by undergraduate Kelsie Bashford.
2Much of the experimental work was done by undergraduates Ryan Simmons and Stephen Burley as well as our lab manager Linda Rogers.
Loftie-Eaton, W., and D. E. Rawlings. 2009. Comparative biology of two natural variants of the IncQ-2 family plasmids, pRAS3.1 and pRAS3.2. J Bacteriol 191:6436-6446.
Loftie-Eaton, W., and D. E. Rawlings. 2010. Evolutionary competitiveness of two natural variants of the IncQ-like plasmids, pRAS3.1 and pRAS3.2. J Bacteriol 192:6182-6190.
Kado, C. I. 1998. Origin and evolution of plasmids. Antonie Van Leeuwenhoek 73:117-126.
Smillie, C., M. P. Garcillan-Barcia, M. V. Francia, E. P. Rocha, and F. de la Cruz. 2010. Mobility of plasmids. Microbiol Mol Biol Rev 74:434-452.
Stewart, F. M., and B. R. Levin. 1977. The population biology of bacterial plasmids: a priori conditions for the existence of conjugationally transmitted factors. Genetics 87:209-228.
Ponciano, J. M., L. De Gelder, E. M. Top, and P. Joyce. 2007. The population biology of bacterial plasmids: a hidden Markov model approach. Genetics 176:957-968.
Bergstrom, C. T., M. Lipsitch, and B. R. Levin. 2000. Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155:1505-1519.
Bouma, J. E., and R. E. Lenski. 1988. Evolution of a bacteria/plasmid association. Nature 335:351-352.
Sota, M., H. Yano, H. M, Julie, G. W. Daughdrill, Z. Abdo, L. J. Forney, and E. M. Top. 2010. Shifts in the host range of a promiscuous plasmid through parallel evolution of its replication initiation protein. ISME J 4:1568-1580.
Dahlberg, C., and L. Chao. 2003. Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12. Genetics 165:1641-1649.
Loftie-Eaton, W., Burleigh, S., Simmons, R., Rogers, L., Hunter, S., Settles, M., Ponciano, J.M. and Eva Top. Transposition of a toxin-antitoxin system and plasmid-host coevolution increase plasmid persistence and host range. In preparation.
For more information about Wesley’s work, you can contact him at wesleyl at uidaho dot edu.
Wiki hii's BEACON Watafiti katika Kazi blog post ni na Chuo Kikuu cha Idaho postdoc Wesley Loftie-Eaton. Nilijifunza kuhusu plasmids katika chuo kikuu cha Stellenbosch nchini Afrika Kusini. Mshauri wangu, Profesa D. E. Rawlings, aliniomba kuamua athari za kibiolojia za mabadiliko maalum sana ndani ya asili ya replication ya plasmids mbili isogenic (Lofti-Eaton na Rawlings, 2009). Swali hilo lililoanza kuwa rahisi na la moja kwa moja lilinivutia upesi kwa sababu ya utata wake wa kweli. Wakati huo, nilijifunza kwamba huwezi kuelewa plasmids, au kitu chochote katika biolojia kwa jambo hilo, bila muktadha wa mageuzi; na ni mahali gani bora pa kujifunza mageuzi ya plasmid kuliko katika Chuo Kikuu cha Idaho pamoja na Dk Eva M. Top na chini ya mwavuli wa IBEST na BEACON. Katika kesi wewe si plasmid geek kama mimi, plasmids ni kwa ujumla mviringo (maumbo linear pia zipo) DNA vyombo kwamba kuiga kujitegemea katika bakteria. Plasmids ndogo zaidi zina pairi za msingi mia chache, wakati kubwa zaidi zina pairi za msingi zaidi ya milioni moja (Kado et al., 1998). Kulingana na habari ya mfululizo, karibu nusu ya plasmids ni au kujihamisha kwa njia ya conjugation au inaweza kuhamishwa (kuhamishwa) na plasmids conjugative, wakati wengine kuonekana kuwa non-transferable. Plasmids nyingi ni siri au hazina jeni yoyote isiyo muhimu wakati wengine wanaweza kubeba jeni ambazo hutoa faida kwa mwenyeji wa bakteria, ikiruhusu kuchukua na kuenea katika niche isiyo ya uhasama (Kado et al., 1998). Kulingana na kama plasmid hutoa mwenyeji wake na faida au gharama, wanaweza kulinganishwa na symbionts au vimelea, kwa mtiririko huo, na kama symbionts na vimelea, wana safu ya mwenyeji ambayo ni nyembamba au pana. "Mipango ambayo ninafanya kazi sasa inazingatia ""kufafanua utaratibu wa molekuli ambao huwezesha plasmids kubadilisha, kukata au kupanua safu yao ya mwenyeji"" na ""kuelewa jinsi plasmids ya safu pana ya mwenyeji wanavyoongezeka na kuenea katika jamii ngumu za vijidudu.""" Jambo hilo la mwisho halitajadiliwa hapa. Mbali na asili ya msingi ya maswali haya, ni muhimu kwa afya ya binadamu kutokana na kuenea kwa plasmid-mediated ya upinzani wa antibiotic kati ya vimelea. Wakati plasmid conjugates kwa mwenyeji naive inaweza si lazima kudumu isipokuwa kuna uteuzi kwa ajili ya matengenezo yake katika mwenyeji kwamba. Kushindwa kudumu kunaweza kuwa kwa sababu ya replication isiyo na ufanisi, kugawanyika vibaya au kupoteza kwa plasmid wakati wa mgawanyiko wa seli, au seli zenye plasmid zinaweza tu kushindwa na seli zisizo na plasmid ikiwa plasmid inaweka gharama kwa mwenyeji. Plasmid na mwenyeji wanaweza kubadilika kwa haraka wakati wa uteuzi mkubwa wa plasmid, baada ya hapo plasmid inaweza kuendelea kwa muda mrefu hata wakati uteuzi mkubwa umeondolewa (Bouma na Lenski, 1988; Dahlberg na Chao, 2003; Sota et al., 2010). Kwa mfano, katika picha ya chini, kuna uhusiano wa plasmid-mchungaji ambao haukuwa imara, na baada ya vizazi 200 hivi, phenotype imebadilika na baada ya vizazi 600 hivi, plasmid ilikuwa imara sana katika mwenyeji. "Hata hivyo, haikuwa wazi kutoka kwa takwimu hii (kwa sababu mimi si kuonyesha data kamili ya kipimo) kwamba katika kukosekana kwa uteuzi wa antibiotic, plasmid ya mababu ilisonga kuelekea kutoweka, hata hivyo, uhusiano wa kudumu ambao plasmid ilihifadhiwa katika ~ 10% ya idadi ya watu kutokana na uhamisho wa usawa uliibuka ndani ya vizazi vya kwanza 100 (paneli C, chini).""" Pamoja na washirika Sam Hunter na Haruo Suzuki, mimi sasa ninafanya kazi kuelezea mabadiliko ya maumbile nyuma ya ongezeko hili la uvumilivu, na mshirika Jose Ponciano anafanya kazi kwa njia ya kupima jinsi rahisi au ngumu ni kubadili kati ya trajectories ya uvumilivu na kutoweka. Katika jaribio jingine (plasmid tofauti na mwenyeji) tuligundua kwamba transposon encoded juu ya plasmid asili kwa mwenyeji kwamba <unk>jumped<unk> kwa plasmid kuletwa, ambayo ilikuwa kudumishwa chini ya uteuzi wa antibiotic. Matokeo yalikuwa kuongezeka kwa utulivu wa plasmid iliyowekwa, hata bila uteuzi wa antibiotic, na kupoteza plasmid ya asili. Transposon ina mfumo wa toxins na antitoxins (TA) na resolvase, ambayo yote mawili yameonyeshwa kukuza utulivu wa plasmid. Hata hivyo, plasmid iliyobadilika ambayo ilipata transposon ilikuwa imara kabisa katika beta- na gamma-proteobacteria nyingine ambazo babu yake hakuwa imara. Hivyo, upatikanaji wa transposon encoding utulivu kazi ilisababisha kupanuka dhahiri ya plasmid ya mwenyeji mbalimbali na kwa ujumla zaidi, tulionyesha mwingiliano na hatima ya jeni ambayo inaweza arguably kuwa lebo kama <unk> selfish <unk>. Katika mfumo mwingine moja ya matokeo ilikuwa mutation kufuta ndani ya plasmid ya asili ya replication. Ingawa bado kuna kazi nyingi ya kufanywa, badiliko hili limenifurahisha sana. Aina na eneo la mabadiliko ni sawa na mabadiliko niliyojiwekea kuelewa wakati wa PhD yangu. "Kitu cha kushangaza ni kwamba plasmids nilizochunguza wakati huo zilikuwa za familia tofauti na zilitokea katika mazingira, wakati hapa mabadiliko yalitokea wakati wa mageuzi ya majaribio katika maabara.""" Matokeo ya awali yalionyesha kwamba kuondolewa kwa plasmid hii iliondoa uwezo wa kuiga katika mwenyeji wa awali, ambayo tayari ni habari mpya, hata hivyo, ikiwa nadharia yangu kulingana na utafiti wangu wa awali inazuia uchunguzi, basi matokeo haya yatathibitisha kwamba kile tunachokiona wakati wa mageuzi ya majaribio katika maabara pia hutokea katika asili, na kinyume chake. Uthibitisho! Ingawa ni mbali na kamili, kile tumejifunza hadi sasa ni kwamba plasmid mwenyeji-range inaweza kubadilika kwa kasi sana, kwamba mabadiliko kama hayo ya haraka huwa na kutokea kupitia mutations kwamba kusababisha faida au hasara ya kazi na kwamba kuna suluhisho nyingi molekuli ambayo inaweza kusababisha imara plasmid-mwalimu mahusiano. Kutoka kwa mtazamo wa geek ya plasmid hii ni ya kuvutia, lakini kutoka kwa mtazamo wa matibabu ni ya wasiwasi; mara moja plasmid ya upinzani wa dawa nyingi imeanzishwa katika idadi ya watu inakusudia kukaa, hata kama viuavijasumu ambavyo awali vimechaguliwa kwa ajili ya matengenezo yake huondolewa kutoka kwa mfumo! Hata hivyo, kwa kukusanya data zaidi ya aina hii tunatumaini kufafanua mifumo ya jumla katika mageuzi ya plasmid mwenyeji mbalimbali ambayo inaweza kusaidia katika maendeleo ya dawa mpya kuzuia kuenea na kuanzishwa kwa plasmids multidrug upinzani. Majaribio hayo yalifanywa na mwanafunzi wa chuo kikuu Kelsey Bashford. Kazi nyingi za majaribio zilifanywa na wanafunzi wa shahada ya kwanza Ryan Simmons na Stephen Burley pamoja na meneja wetu wa maabara Linda Rogers. Loftie-Eaton, W., na D. E. Rawlings. Mwaka 2009 Biolojia ya kulinganisha ya aina mbili za asili za familia ya plasmids ya IncQ-2: pRAS3 na pRAS3. J Bacteriol 1916436-6446 Loftie-Eaton, W., na D. E. Rawlings. Mwaka 2010 Uwezekano wa kuenea kwa aina mbili za plasmid za IncQ-like ni sawa na pPAS3.1. J Bacteriol 192182 6190. Kado, C. I. Mwaka 1998 Asili na mageuzi ya plasmids. Antonie van Leeuwenhoek, 73 Smillie, C., M. P. Garcillan-Barcia, M. V. Francia, E. P. Rocha, na F. de la Cruz. Mwaka 2010 Uhamisho wa plasmids. Biol.Mol.Biol.Rev.7434-452 Stewart, F. M., na B. R. Levin. Mwaka wa 1977. Biolojia ya idadi ya plasmids bakteria: hali ya a priori kwa ajili ya kuwepo kwa sababu conjugally kuambukizwa. Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics 8720228: Genetics Ponciano, J. M., L. De Gelder, E. M. Top, na P. Joyce. Mwaka wa 2007. Biolojia ya idadi ya plasmids bakteria: siri Markov mfano mbinu. Genetics 176:957-968 Bergstrom, C. T., M. Lipsitch, na B. R. Levin. Mwaka 2000. Uchaguzi wa asili, uhamisho wa maambukizi na hali ya kuwepo kwa plasmids ya bakteria. Genetics 155-1519 Bouma, J. E., na R. E. Lenski. Mwaka 1988 Mageuzi ya bakteria-plasmid ushirika. Utamaduni 335.51-352. Sota, M., H. Yano, H. M., Julie, G. W. Daughdrill, Z. Abdo, L. J. Forney, na E. M. Top. Mwaka 2010 Mabadiliko katika wigo wa mwenyeji wa plasmid promiscuous kupitia mageuzi sambamba ya protini yake ya kuanzisha replica. JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI: JIMI Dahlberg, C., na L. Chao. Mwaka 2003 Uwezekano wa kuhifadhi plasmid katika mimea ya Escherichia coli Genetics 165-1649 Loftie-Eaton, W., Burleigh, S., Simmons, R., Rogers, L., Hunter, S., Settles, M., Ponciano, J. M. na Eva Juu. Transposition ya mfumo wa sumu-antitoxin na coevolution plasmid-mwalimu kuongeza plasmid uvumilivu na wigo mwenyeji. Katika maandalizi. Kwa habari zaidi kuhusu kazi ya Wesley, unaweza kuwasiliana naye katika wesleyl katika uidaho dot edu.