Vpliv sorodstvene diskriminacije na komplementacijo skupnih dobrin v mešani kulturi Bacillus subtilis

Butolen, Monika (author), Mandić Mulec, Ines (mentor), Kraigher, Barbara (co-mentor)

Po Gramu pozitivne paličaste bakterije Bacillus subtilis so sposobne hitrega in koordiniranega skupinskega gibanja po poltrdni površini, imenovanega rojenje. Bakterije za uspešno rojenje potrebujejo bičke in surfaktante. Surfaktant surfaktin B. subtilis izloča v okolje in ga zato uvrščamo med skupne dobrine (ang. »public goods«), ki jih lahko koristijo drugi, tudi goljufi, ki ne prispevajo v skupno dobro. Ločevanje med bolj in manj sorodnimi sevi B. subtilis omogoča sorodstvena diskriminacija. Nesorodni sevi (»non-kin«) rojijo ločeno po površini, medtem ko se sorodni (»kin«) združujejo v skupen roj, kar bi lahko omejilo navzkrižno izkoriščanje skupnih dobrin. Ni znano, ali sorodstvena diskriminacija vpliva na medsebojno delitev skupne dobrine, kot je surfaktin, kar smo preučevali v tej magistrski nalogi. V ta namen smo pripravili rekombinantne seve, označene s fluorescenčnimi markerji in s fluorescenčno stereomikroskopijo ovrednotili uspešnost rojenja dveh sevov, ki smo jih nacepili skupaj na sredino agariziranega gojišča B. Ugotovili smo, da si sorodni sevi lahko med seboj učinkovito delijo surfaktin, saj producent surfaktina omogoči rojenje mutanti srfAA in skupaj z njo zasede celotno površino agarja. Pri nesorodnih sevih je bila komplementacija izrazito manj uspešna zaradi močnega antagonizma med sevi, zato je površino največkrat zasedel le en sev. Uspešnost zasedanja površine je bila odvisna od števila celic posameznega seva v začetnem inokulumu in preživetja napada drugega seva. Pokazali smo tudi, da lahko izrabljeno gojišče nesorodnega seva omogoči rojenje mutanti srfAA. Prav tako so mutante hag, ki nimajo bičkov in zato ne rojijo, delile svoj surfaktin z mutantami srfAA. Pri sorodnih sevih je komplementacija zelo učinkovita, pri nesorodnih je možna le, kadar oba seva preživita in sta prostorsko ločena na plošči. Medsebojna delitev surfaktina je torej učinkovita med sorodnimi sevi, med nesorodnimi pa je delitev te skupne dobrine neučinkovita zaradi medsebojnega izključevanja sevov. Razlike med učinkovitostjo surfaktina, ki ga izloča sorodni ali nesorodni sev, nismo zaznali. Zaključimo lahko, da je sorodstvena diskriminacija zelo pomembna pri omejevanju izrabljanja skupnih dobrin med nesorodnimi sevi.

• Language: Slovenian
• Keywords: Bacillus subtilis, rojenje, sorodstvena diskriminacija, skupne dobrine, surfaktin
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Butolen]
• Year: 2018
• PID: 20.500.12556/RUL-103294
• UDC: 579.22/.26:579.852.11
• COBISS.SI-ID: 4959864
• Publication date in RUL: 15.09.2018

Secondary language

• Language: English
• Title: Influence of kin discrimination on the complementation of public goods in mixed culture of Bacillus subtilis

Abstract

Bacillus subtilis are Gram–positive rod shaped bacteria capable of rapid and coordinated group movement over a semi-solid surface, called swarming. Synthesis of flagella and surfactin are essential for successful bacterial swarming. Surfactant surfactin is secreted into the medium and is therefore classified as public good that can be used by others, including cheaters which do not contribute to the production of public goods. It was observed that B. subtillis can distinguish between highly related and less related strains by kin discrimination. Non-kin strains are swarming separately on the surface, while the related kin strains colonize the surface in a common swarm, which could limit the cross-use of public goods. It is not known whether kin discrimination affects the mutual sharing of public goods, such as surfactin, which we have studied in this master's thesis. Recombinant strains labeled with fluorescence proteins were prepared and then mixed and inoculated on B-medium. The surface colonization success of both strains was evaluated by fluorescence stereomicroscopy. We found that kin strains can effectively share surfactin with srfAA mutants and therefore colonize the entire agar surface together. Complementation was significantly less successful between non-kin strains, due to strong antagonism, and therefore the surface was mostly occupied by only one strain. Which strain occupied the surface was dependent on the ratio between the strains in the initial inoculum and the survival of an attack by another strain. We have shown that srfAA mutants can swarm when supplemented by a conditioned medium of a non-kin or kin strain. It was also observed that hag mutants, which do not produce flagella and therefore cannot swarm, share their surfactin with kin srfAA mutants, but with non-kin only when both strains survived and covered the surface area in spatially separated swarms. The sharing of surfactin is effective between related strains, while among less related strains, it is ineffective due to the mutual exclusion of strains. The differences between the effectiveness of surfactin secreted by kin or non-kin strains were not detected. We can conclude that kin discrimination limits the exploitation of public goods among non-kin strains.

• Keywords: Bacillus subtilis, swarming, kin discrimination, public goods, surfactin