When disaster strikes: Reconstitution of population density by expansion of survivors

Microorganisms have an assortment of stress-response mechanisms that enable them to survive in the face of environmental stresses. However, with prolonged exposures to severe stresses adaptive stress responses ultimately fail, the affected populations may suffer a massive decline. Recovery of the population density in the aftermath of a massive death is a vital task. Our recent post-stress regrowth under starvation (RUS) studies prompted us to propose RUS as an adaptation for overcoming consequences of devastating environmental disturbances. RUS should be seen as an integral process having two major aspects: the stress-induced cellular auto-decomposition and the recycling of the released nutrients. Here, we summarized what is already known about RUS and suggest a number of questions that are key to understanding the molecular underpinnings of these two operations. We also interrogate the prospect that would conceptualize the auto-decomposition as a fitness-maximizing mechanism acting with the purpose of an expedient supply of nutrients. Two further things are of special note: given that some of the RUS-defective mutants are also impaired in DNA repair, RUS can serve as an important tool for uncovering new determinants operating, in some overlapping fashion, in the protection of genome integrity; also, RUS can serve as a new angle of approach that might, hopefully, assign roles to some of those (up to similar to 30%) of microbial genes that are of unknown function. More generally, understanding post-stress reconstitution and the underlying mechanisms is a necessary (complementing) part of any comprehensive picture of how microbes cope with very harsh environmental disturbances

Analyses of expression, activity and localization of the new type of aspartic proteinase (FeAPL1) from buckwheat (Fagopyrum esculentum Moench)

Iz biblioteke cDNK semena heljde u srednjoj fazi sazrevanja prethodno je izolovana cDNK koja kodira atipičnu aspartičnu proteinazu (FeAPL1), a zatim i odgovarajući genomski fragment koji, osim kodirajuće sekvence, sadrži i 5’-regulatorni region u kome su uočeni cis-elementi koji mogu učestvovati u regulaciji ekspresije ovog gena pod uticajem faktora spoljašnje sredine i opredeljivati njegovu seme-specifičnu ekspresiju. U ovom radu uradjena je detaljna analiza ekspresije gena FeAPL1 po organima heljde- na nivou RNK upotrebom metoda RT-PCR i Real Time RT-PCR, a analizom Western blot na nivou proteina. Umnošci slabog intenziteta su uočeni u fazama sazrevanja semena do stadijuma 14 DAF, za kojima sledi naglo povećanje količine transkripta u stadijumu 14-17 DAF, nakon čega dolazi do smanjenja količine RNK. Profil ekspresije proteina prati profil ekspresije RNK. U listu, korenu, stablu i cvetu uočene su male količine transkripta, ali odgovarajući protein nije bio prisutan. Kako je kompjuterskom analizom predikovano više cis-aktivirajućih elemenata u promotorskom regionu gena za FeAPL1, njegova ekspresija je praćena i pod uticajem stresogenih faktora spoljašnje sredine (UV zračenje, mrak, mehanička povreda, suša, salicilna kiselina), ali značajne promene u ekspresiji nisu oučene ni u jednom ispitivanom slučaju. U cilju dobijanja proteina u količini pogodnoj za biohemijsku analizu i produkciju antitela, korišćeno je nekoliko ekspresionih sistema: E. coli, P. pastoris i kultura insekatskih ćelija. Zadovoljavajuća ekspresija dobijena je jedino pri upotrebi konstrukta rMBP-FeAPL1 u soju Rosetta-gami E. coli u kome je dobijen solubilan rekombinantni fuzioni proteina koji, nakon proteolitičke obrade, poseduje enzimsku aktivnost na pH 3,0, inhibiranu pepstatinom A. Rekombinantni protein, rFeAPL1-His6, od ~55 kDa, je eksprimiran u ćelijskoj liniji duvana BY-2 u kome je detektovana pepstatin A senzitivna enzimska aktivnost na pH 3,0 bez prethodne proteolitičke obrade. Veća molekulska masa u odnosu na predikovanu (46 kDa) ukazuje na verovatnu glikozilaciju proteina. Imunocitohemijskom analizom i analizom proteina protoplasta transformisanih BY-2 ćelija je utvrđeno da je FeAPL1 lokalizovan u ćelijskom zidu. Na osnovu dobijenih rezultata diskutovane su potencijalne funkcije proteinaze FeAPL1

Self-Generated Hypoxia Leads to Oxidative Stress and Massive Death in Ustilago maydis Populations under Extreme Starvation and Oxygen-Limited Conditions

Ustilago maydis and Saccharomyces cerevisiae differ considerably in their response to water-transfer treatments. When stationary phase cells were transferred to pure water and incubated under limited supply of oxygen, the U. maydis cells suffered a catastrophic loss of viability while the S. cerevisiae population was virtually unaffected by the treatment. The major factor underlying the death of the U. maydis cells under those conditions was an oxygen-consuming cellular activity that generated a hypoxic environment, thereby inducing oxidative stress and accumulation of reactive oxygen species, which resulted in lethality. Importantly, a small residue of U. maydis cells that did survive was able to resume growth and repopulate up to the initial culture density when sufficient aeration was restored. The regrowth was dependent on the cellular factors (Adr1, Did4, Kel1, and Tbp1), previously identified as required for repopulation, after killing with hydrogen peroxide. Surprisingly, the survivors were also able to resume growth under apparently hypoxic conditions, indicating that these remnant cells likely switched to a fermentative mode of growth. We discuss the findings in terms of their possible relevance to the eco-evolutionary adaptation of U. maydis to risky environments

Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis

Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA intermediates that result as a consequence of homologous recombination and distressed replication. Ordinarily the homologous recombination system serves as a high-fidelity mechanism to restore the integrity of a damaged genome, but in the absence of the appropriate resolving function it can turn DNA intermediates resulting from replication stress into pathological forms that are toxic to cells. Here we have investigated how the nucleases Mus81 and Gen1 and the helicase Blm contribute to survival after DNA damage or replication stress in Ustilago maydis cells with crippled yet homologous recombination-proficient forms of Brh2, the BRCA2 ortholog and primary Rad51 mediator. We found collaboration among the factors. Notable were three findings. First, the ability of Gen1 to rescue hydroxyurea sensitivity of dysfunctional Blm requires the absence of Mus81. Second, the response of mutants defective in Blm and Gen1 to hydroxyurea challenge is markedly similar suggesting cooperation of these factors in the same pathway. Third, the repair proficiency of Brh2 mutant variants deleted of its N-terminal DNA binding region requires not only Rad52 but also Gen1. and Mus81. We suggest these factors comprise a sub pathway for channeling repair when Brh2 is compromised in its interplay with DNA.Published version: [https://imagine.imgge.bg.ac.rs/ha]ndle/123456789/1190This is the peer reviewed version of the paper: Kojic, M., Milisavljevic, M., & Holloman, W. K. (2018). Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis. DNA Repair, 63, 47–55.[ https://doi.org/10.1016/j.dnarep.2018.01.010

Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli

U ovom radu predstavljena je ekspresija rekombinantne atipične aspartatne proteinaze heljde (Fagopyrum esculentum) bogate cisteinom, gde su testirana različita ekspresiona svojstva pet sojeva E. coli. Takođe je analiziran i uticaj fuzionih partnera (His6 i MBP) na efikasnost ekspresije. U slučaju His6-FeAPL1, dobijena je velika količina nerastvornog proteina, smeštenog u inkluzionim telima. S druge strane, MBP-FeAPL1 je bio lokalizovan i u citoplazmi i u inkluzionim telima u oba upotrebljena soja E. coli (BL21 i Rosetta-gami). Međutim, samo za rekombinantni protein proizveden u soju Rosetta-gami, dokazana je proteolitička aktivnost na supstratu BSA, pri pH 3,0. Rezultati su takođe ukazali da FeAPL1 sadrži PRO segment, čije je odstranjivanje neophodno za njegovu proteolitičku aktivnost. Aktivnost FeAPL1, pokazana samo u soju Rosetta-gami, gde je moguće formiranje disulfidnih veza, ukazuje na značaj 12 cisteina u uspostavljanju pravilne strukture koja omogućava funkcionalnost enzima.Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality

Identification of Genes Promoting Growth of Ustilago maydis on Biomolecules Released from Cells Killed by Oxidation

Much headway has been made in understanding the numerous strategies that enable microorganisms to counteract various types of environmental stress, but little is known about how microbial populations recover after a massive death caused by exposure to extreme conditions. Using the yeast-like fungus Ustilago maydis as a model, our recent post-stress regrowth under starvation (RUS) studies have demonstrated that this organism reconstitutes devastated populations with remarkable efficiency. Subsequently, we have identified four RUS-gene products. Two of these, Did4 and Tbp1, play parallel roles in protecting the genome. To identify additional molecular components, we took a molecular-genetic and a transcriptomic approach. By employing a simple and novel screening method, we identified five RUS-deficient mutants (snf8, slm1, vrg4, snf5, hsf1), three of which (snf8, slm1, and hsf1) displayed sensitivity to different genotoxic agents, indicating that the corresponding gene products have roles in genome protection. The global transcriptomic changes of cells grown in supernatants derived from peroxide-treated cell suspensions revealed sets of uniquely expressed genes. Importantly, among the genes induced by the substrates was Chk1, which encodes a protein kinase required for checkpoint-mediated cell cycle arrest in response to DNA damage. Mutants of U. maydis deleted of Chk1 are severely incapacitated in RUS

NOVEL CELLULAR FACTORS INVOLVED IN REGULATION OF BRCA2-DRIVEN HOMOLOGOUS RECOMBINATION IN USTILAGO MAYDIS

Homologous recombination (HR) is one of the most accurate mechanisms of preserving genome integrity by precise repair of double strand breaks as the most deleterious type of DNA damage. Mechanisms of HR are mostly studied in yeast which, unlike higher eukaryotes employs RAD52 as a HR mediator, instead of BRCA2. Ustilago maydis is a unicellular phytopathogen characterized by extreme radiation resistance dependent on BRCA2-driven HR. The focus of our research is to uncover novel cellular factors that regulate HR, by isolating suppressors of blm in U. maydis. We have identified 3 new factors of unknown functions, as well as Rad55 and Mph. All mutations suppress HU sensitivity of blm. Presence of truncated proteins caused by point mutations that introduce the specific premature STOP codon and complete deletions of UMAG_01566 and UMAG_01667, both lead to genotoxins sensitivity and altered growth rates on HU to a various extent. Mutation in UMAG_03150 leads to slow growth which can be suppressed by truncated UMAG_01566. Mitotic or meiotic recombination is also affected in some of the mutants. We assume that these novel factors can provide insights into HR regulation, interactions among HR participants and relations to other cellular processes.Fusion Conferences:Recombination Mechanisms 10-13 July 2023, Lisbon, Portuga

Two metallothionein gene family members in buckwheat: Expression analysis in flooding stress using Real Time RT-PCR technology

Metalotioneini (MT) pripadaju velikoj grupi proteina male molekulske težine bogatih cisteinom, izražene sposobnosti za vezivanje jona metala, uključenih u procese održavanja homeostaze metalnih jona i detoksifikacije od teških metala. U radu je analizirana struktura dva transkripta gena za MT tipa 3 poreklom iz semena heljde u razviću. Razlike su nađene pre svega u okviru 3’- UTR sekvenci. Nakon analiza sekvenci urađena je analiza ekspresije tokom hipoksije korišćenjem tehnologije Real Tme RT-PCR.Metallothioneins (MTs) are an extensive and diverse family of small cysteine-rich proteins with metal-binding ability that are involved in metal homeostasis and detoxification. Two cDNA clones of the MT3 type, differing in 3’ UTRs, were isolated from the developing buckwheat seed cDNA library. Following sequence analyses, expression profiles during flooding stress were monitored by Real Time RT PCR technology

Нови ћелијски фактори у регулацији BRCA2-зависне рекомбинације у Ustilago maydis

Održavanje stabilnosti genoma je jedna od najvažnijih ćelijskih funkcija, koja joj omogućava preživljavanje i deobu. Jedan od mehanizama popravke DNK je homologna rekombinacija (HR), čija mašinerija omogućava preciznu popravku dvolančanih prekida, kao najštetnijih oštećenja DNK, te narušavanje HR može dovesti do genomske nestabilnosti, koja je u osnovi čitavog niza bolesti. Uprkos opsežnim istraživanjima HR na različitim organizmima i dalje nisu detaljno okarakterisane sve funkcije poznatih HR-faktora, a otkrivaju se i novi faktori uključeni u ovaj proces. Kao pogodan model sistem za pronalaženje novih ćelijskih faktora HR-e, koristimo jednoćelijsku bazidiomicetu Ustilago maydis, koja je visoko rezistentna na zračenje, poseduje osnovnu HR-mašineriju sličnu humanoj, sa konzerviranim BRCA2 ortologom (Brh2)1 i ima 30% gena nepoznate funkcije. Dizajniran je specifični molekularno-genetički pristup – kroz izolovanje supresora mutanta blm, što zapravo znači izolovanje funkcionalnih partnera Blm helikaze, čija je aktivnost sinhronizovana sa procesom popravke DNK putem HR.2 Do sada su izolovana tri nova supresora (bls2, bls9 i bls10), generisani su pojedinačni mutanti i okarakterisan je njihov fenotip. Identifikovani geni kodiraju hipotetičke proteine. Bls9 i bls10 su izrazito senzitivni na genotoksične agense i imaju narušenu efikasnost HR, dok bls2 ima usporen rast. Detaljnom molekularno-genetičkom i biohemijskom analizom će se definisati uloge ovih faktora u HR.Одржавање стабилности генома је једна од најважнијих ћелијских функција, која јој омогућава преживљавање и деобу. Један од механизама поправке ДНК је хомологна рекомбинација (ХР), чија машинерија омогућава прецизну поправку дволанчаних прекида, као најштетнијих оштећења ДНК, те нарушавање ХР може довести до геномске нестабилности, која је у основи читавог низа болести. Упркос опсежним истраживањима ХР на различитим организмима и даље нису детаљно окарактерисане све функције познатих ХР-фактора, а откривају се и нови фактори укључени у овај процес. Као погодан модел систем за проналажење нових ћелијских фактора ХР-е, користимо једноћелијску базидиомицету Ustilago maydis, која је високо резистентна на зрачење, поседује основну ХР-машинерију сличну хуманој, са конзервираним BRCA2 ортологом (Brh2)1 и има 30% гена непознате функције. Дизајниран је специфични молекуларно-генетички приступ – кроз изоловање супресора мутанта blm, што заправо значи изоловање функционалних партнера Blm хеликазе, чија је активност синхронизована са процесом поправке ДНК путем ХР.2 До сада су изолована три нова супресора (bls2, bls9 и bls10), генерисани су појединачни мутанти и окарактерисан је њихов фенотип. Идентификовани гени кодирају хипотетичке протеине. Bls9 и bls10 су изразито сензитивни на генотоксичне агенсе и имају нарушену ефикасност ХР, док bls2 има успорен раст. Детаљном молекуларно-генетичком и биохемијском анализом ће се дефинисати улоге ових фактора у ХР.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022

Isolation and structural analysis of a gene coding for a novel type of aspartic proteinase from buckwheat seed (Fagopyrum esculentum Moench)

Iz biblioteke cDNK semena heljde u srednjoj fazi razvića izolovan je gen koji kodira novi tip aspartične proteinaze. Analizom sekvence ove cDNK (Fe-APL1) uočeno je odsustvo domena karakterističnog samo za biljne aspartične proteinaze, a analizom odgovrajućeg genomskog fragmenta da se radi o genu koji ne sadrži introne. Bioinformatičkim analizama genoma arabidopsisa je pokazano da je većina potencijalnih gena za aspartične proteinaze upravo sa ovim osobinama, iako je to eksperimentalno dokazano samo kod malog broja gena. Rezultati ovog rada daju doprinos u analizi raznovrsnosti unutar familije biljnih aspartičnih proteinaza. .A novel type of aspartic proteinase gene was isolated from the cDNA library of developing buckwheat seeds. This cDNA, FeAPL1, encoded an AP-like protein lacking the plant-specific insert (PSI) domain characteristic of typical plant aspartic proteinases. In addition the corresponding genomic fragment was isolated. It is demonstrated that this gene does not contain introns. Since bioinformatics analysis of the Arabidopsis genome showed that most potential AP genes are intronless and PSI-less, it appears that "atypical" is an inappropriate word for that class of AP. Isolation of this specific buckwheat gene among the small group of those isolated from other plant species provides a new perspective on the diversity of AP family members in plants.