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

BRCA2-RAD51-DSS1 interplay examined from a microbial perspective

The tumor suppressor BRCA2 plays an essential role in repair of double-strand DNA breaks by regulating the action of the RAD51 recombinase. The activity of BRCA2 in turn is governed by DSS1, a small acidic protein that appears to function as a necessary cofactor. A model fungal system that reproduces the BRCA2-RAD51 interaction offers the opportunity to understand at the molecular level the mechanism of DSS1 activation

Analysis of regulatory regions and expression of the sgm gene from Micromonospora zionensis

Proizvodjac aminoglikozidnog antibiotika G-52, Micromonospora zionensis, poseduje sgm gen koji kodira 16$ rRNK metilazu cijom se ak'uvnoscu ostvaruje rezistencija na 4,6-disupstituisane dezoksistreptaminske aminoglikozide. Identifikovana su dva promotora uzvodno od kodirajuce sekvence sgm gena. Jedan promoter je reiativno slab i on otpocinje transkripciju od G nukleotida koji se nalazi 72 bp uzvodno od translacionog start kodona (ATG). Drug: promotor je mnogo jaci i Iociran je na vecoj udaljenosti od ATG kodona (~250 nukleotida). Postojanje ovih tandemskih promotora bi moglo da omoguci diferencijalnu gensku ekspresiju pa je postulirana njihova razlicita funkcija kod proizvodjaca. Tako 1e predlozeno da je slabiji P1 promoter odgovoran za konstitutivnu transkripciju sgm gena, dok je P2 zaduzen za ekspresiju nizvodnih (biosintetskih) gena u vreme kada otpocinje sinteza antibiotika. Prema tome, P2 promotor bi mogao biti potencijalno koristan za favorizovanu ekspresiju kloniranih gena u stacionarnoj fazi rasta micromonospora. Konstruisana je serija transkripcionih i translacionih sgm-Iacz genskih fuzija na plazmidu pPLti7G. Transkripcija obe vrste fuzija se odvija pod kontrolom snaznog PLtI promotora, posto promotori sgm gena nisu funkcionalni u bakteriji E. coli. Nivo ekspresiie lacZ gena u pojedinim transformantima sa transkripcionim ili translacionim fuzijama odredjivan je merenjem B-galaktozidazne aktivnosti. Transformanti sa translacionom fuznjom koji imaju i ekstra kopiju sgm gena bilo u cis iii u trans poziciji, pokazuju izrazit pad B-galaktozidazne aktivnosti. U slicnim eksperimentima sa transkripcionim fuzijama nije uocen znacajan efekat ekstra kopije sgm gena. Ovi rezultati su pokazali da je ekspresija sgm gena autoregulisana transiacionom represijom, najverovatnije zbog vezivanja metilaze za sopstvenu iRNK. Ekspresijom sgm gena u razlicitim sojevima aktinomiceta utvrdjeno je da je visok nivo rezistenqije na higromicin B zaista determinisan sgm genom, ali da je ispoljavanje ove rezistencije zavisno od soja u kome se sgm gen eksprimira. Ovaj podatak sugerira na zakljucak da ispitivani sojevi mikromonospora poseduju identican mehanizam rezistencije na higromicin. Iz toga se moze zakljuciti da mikromonospore najverovatnije poseduju odredjenu specificnost u gradji male subjedinice ribozoma koja je. konzervirana medju sojevima ovog roda.The sisomicin-gentamicin resistance methylase (sgm) gene from Micromonospora zionensis (producer of 6-52antibiotic), encodes an enzyme thatmodifies 16S rRNA, and thereby conferring resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Two promoters were identified upstream of the sgm coding sequence. One promoter (Pi) is relatively weak and initiates transcription at the G which is 72 nucleotides upstream of the translational initiation codon (ATG). A second (P2) promoter is much stronger and is located more upstream from the ATG codon (~250 nucleotides). These tandem promoters could enable differential gene expression of the sgm gene, and it has been postulated that promoters have specialized functions in the producing organism. The apparent weak P1 promoter is probably responsible for constitutive transcription of the sgm gene, whereas the P2 promoter may play a specialized role in expressron of the downstream (biosynthetic) genes, during the time that the antibiotic (3-52 is produced. Therefore, P2 promoter could be potentially used in expressing cloned genes especially during the stationary growth phase of micromonospora. A set of transcriptional and translational fusions oi the sgm gene and the feel reporter gene have been constructed by using the pPLtl7G plasmid. Both transcriptional and translational fusions were transcribed under the strong PLtl promoter,since it has been shown that the sgm promoters are non-functional in E. coli. Transformants harbouring either the transcriptional and translational fusions were assayed for B-gaiactosidase activity. Translational fusion transformants which also carried an extra copy of sgm gene either in cis or in trans position, exhibited a substantial decrease in B-galactosidase activity. No significant effect was observed in comparable experiments with the transcriptional fusions. These results demonstrate that the expression of the sgm gene is regulated by translational autorepression, presumably due to methylase binding to the specific site/s on its own mFiNA. The translational repression model is discussed in light of overall control of the sgm gene expression. The expression of the sgm gene in various actinomycetes strains revealed that expression of high-level resistance to hygromycin B, determined by sgm gene, is background dependent. This evidence suggests that it may well be that ail tested micromonospora strains share a common mode of hygromycin B resistance. Factors that might be contributed to background-dependent expression of hygromycin B resistance are discussed

Brh2 domain function distinguished by differential cellular responses to DNA damage and replication stress

Mutants of the fungus Ustilago maydis defective in the RecQ helicase Blm are highly sensitive to killing by the DNA replication stressor hydroxyurea. This sensitivity or toxicity is dependent on the homologous recombination (HR) system and apparently results from formation of dead-end HR DNA intermediates. HU toxicity can be suppressed by deletion of the gene encoding Brh2, the BRCA2 orthologue that serves to regulate HR by mediating Rad51 filament formation on single-stranded DNA. Brh2 harbours two different DNA-binding domains that contribute to HR function. DNA-binding activity from a single domain is sufficient to provide Brh2 functional activity in HR, but to enable HU-induced killing two functional DNA-binding domains must be present. Despite this stringent requirement for dual functioning domains, the source of DNA-binding domains is less critical in that heterologous domains can substitute for the native endogenous ones. The results suggest a model in which the nature of the DNA lesion is an important determinant in the functional response of Brh2 action.This is the peer reviewed version of the paper: Kojic, M., & Holloman, W. K. (2012). Brh2 domain function distinguished by differential cellular responses to DNA damage and replication stress. Molecular Microbiology, 83(2), 351–361.[ https://doi.org/10.1111/j.1365-2958.2011.07935.x]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/584

Disruptions of the Ustilago maydis REC2 gene identify a protein domain important in directing recombinational repair of DNA

The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was first identified in a screen for UV-sensitive mutants. The original isolate, rec2-1, was found to be deficient in repair of DNA damage, genetic recombination and meiosis. We report here that the rec2-197 allele, which was constructed by gene disruption, retains some biological activity and is partially dominant with respect to REC2. The basis for the residual activity is probably as a result of expression of a diffusible product from the rec2-197 allele that augments or interferes with REC2 functions. This product appears to be a polypeptide expressed from a remnant of the 5' end of the open reading frame that was not removed in creating the gene disruption. The mutator activity and disturbed meiosis of rec2-197 suggest that the Rec2 protein functions in a process that avoids spontaneous mutation and insures faithful meiotic chromosome segregation. A prediction based on the phenotype of rec2-197 is that Rec2 protein interacts with one or more other proteins in directing these functions. To identify interacting proteins we performed a yeast two-hybrid screen and found Rad51 as a candidate. Rec2-197 and Rad51 appear to interact to a similar degree

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

Analysis of regulatory regions and expression of the sgm gene from Micromonospora zionensis

Proizvodjac aminoglikozidnog antibiotika G-52, Micromonospora zionensis, poseduje sgm gen koji kodira 16$ rRNK metilazu cijom se ak'uvnoscu ostvaruje rezistencija na 4,6-disupstituisane dezoksistreptaminske aminoglikozide. Identifikovana su dva promotora uzvodno od kodirajuce sekvence sgm gena. Jedan promoter je reiativno slab i on otpocinje transkripciju od G nukleotida koji se nalazi 72 bp uzvodno od translacionog start kodona (ATG). Drug: promotor je mnogo jaci i Iociran je na vecoj udaljenosti od ATG kodona (~250 nukleotida). Postojanje ovih tandemskih promotora bi moglo da omoguci diferencijalnu gensku ekspresiju pa je postulirana njihova razlicita funkcija kod proizvodjaca. Tako 1e predlozeno da je slabiji P1 promoter odgovoran za konstitutivnu transkripciju sgm gena, dok je P2 zaduzen za ekspresiju nizvodnih (biosintetskih) gena u vreme kada otpocinje sinteza antibiotika. Prema tome, P2 promotor bi mogao biti potencijalno koristan za favorizovanu ekspresiju kloniranih gena u stacionarnoj fazi rasta micromonospora. Konstruisana je serija transkripcionih i translacionih sgm-Iacz genskih fuzija na plazmidu pPLti7G. Transkripcija obe vrste fuzija se odvija pod kontrolom snaznog PLtI promotora, posto promotori sgm gena nisu funkcionalni u bakteriji E. coli. Nivo ekspresiie lacZ gena u pojedinim transformantima sa transkripcionim ili translacionim fuzijama odredjivan je merenjem B-galaktozidazne aktivnosti. Transformanti sa translacionom fuznjom koji imaju i ekstra kopiju sgm gena bilo u cis iii u trans poziciji, pokazuju izrazit pad B-galaktozidazne aktivnosti. U slicnim eksperimentima sa transkripcionim fuzijama nije uocen znacajan efekat ekstra kopije sgm gena. Ovi rezultati su pokazali da je ekspresija sgm gena autoregulisana transiacionom represijom, najverovatnije zbog vezivanja metilaze za sopstvenu iRNK. Ekspresijom sgm gena u razlicitim sojevima aktinomiceta utvrdjeno je da je visok nivo rezistenqije na higromicin B zaista determinisan sgm genom, ali da je ispoljavanje ove rezistencije zavisno od soja u kome se sgm gen eksprimira. Ovaj podatak sugerira na zakljucak da ispitivani sojevi mikromonospora poseduju identican mehanizam rezistencije na higromicin. Iz toga se moze zakljuciti da mikromonospore najverovatnije poseduju odredjenu specificnost u gradji male subjedinice ribozoma koja je. konzervirana medju sojevima ovog roda.The sisomicin-gentamicin resistance methylase (sgm) gene from Micromonospora zionensis (producer of 6-52antibiotic), encodes an enzyme thatmodifies 16S rRNA, and thereby conferring resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Two promoters were identified upstream of the sgm coding sequence. One promoter (Pi) is relatively weak and initiates transcription at the G which is 72 nucleotides upstream of the translational initiation codon (ATG). A second (P2) promoter is much stronger and is located more upstream from the ATG codon (~250 nucleotides). These tandem promoters could enable differential gene expression of the sgm gene, and it has been postulated that promoters have specialized functions in the producing organism. The apparent weak P1 promoter is probably responsible for constitutive transcription of the sgm gene, whereas the P2 promoter may play a specialized role in expressron of the downstream (biosynthetic) genes, during the time that the antibiotic (3-52 is produced. Therefore, P2 promoter could be potentially used in expressing cloned genes especially during the stationary growth phase of micromonospora. A set of transcriptional and translational fusions oi the sgm gene and the feel reporter gene have been constructed by using the pPLtl7G plasmid. Both transcriptional and translational fusions were transcribed under the strong PLtl promoter,since it has been shown that the sgm promoters are non-functional in E. coli. Transformants harbouring either the transcriptional and translational fusions were assayed for B-gaiactosidase activity. Translational fusion transformants which also carried an extra copy of sgm gene either in cis or in trans position, exhibited a substantial decrease in B-galactosidase activity. No significant effect was observed in comparable experiments with the transcriptional fusions. These results demonstrate that the expression of the sgm gene is regulated by translational autorepression, presumably due to methylase binding to the specific site/s on its own mFiNA. The translational repression model is discussed in light of overall control of the sgm gene expression. The expression of the sgm gene in various actinomycetes strains revealed that expression of high-level resistance to hygromycin B, determined by sgm gene, is background dependent. This evidence suggests that it may well be that ail tested micromonospora strains share a common mode of hygromycin B resistance. Factors that might be contributed to background-dependent expression of hygromycin B resistance are discussed

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

Role of Blm and collaborating factors in recombination and survival following replication stress in Ustilago maydis

Inactivation of the structural gene for the RecQ family member, BLM in human, Sgs1 in budding yeast, or Rqh1 in fission yeast leads to inappropriate recombination, chromosome abnormalities, and disturbed replication fork progression. Studies with yeasts have demonstrated that auxiliary gene functions can contribute in overlapping ways with Sgs1 or Rqh1 to circumvent or overcome lesions in DNA caused by certain genotoxic agents. In the combined absence of these functions, recombination-mediated processes lead to severe loss of fitness. Here we performed a genetic study to determine the role of the Ustilago maydis Blm homolog in DNA repair and in alleviating replication stress. We characterized the single mutant as well as double mutants additionally deleted of genes encoding Srs2, Fbh1, Mus81, or Exo1. Unlike yeasts, neither the blm srs2, blm exo1. nor blm mus81 double mutant exhibited extreme loss of fitness. Inactivation of Brh2, the BRCA2 homolog, suppressed toxicity to hydroxyurea caused by loss of Blm function. However, differential suppression by Brh2 derivatives lacking the canonical DNA-binding region suggests that the particular domain structure comprising this DNA-binding region may be instrumental in promoting the observed hydroxyurea toxicity.This is the peer reviewed version of the paper: Mao, N., Kojić, M., & Holloman, W. K. (2009). Role of Blm and collaborating factors in recombination and survival following replication stress in Ustilago maydis. DNA Repair, 8(6), 752–759. [https://doi.org/10.1016/j.dnarep.2009.02.006]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/343

BRCA2 homolog required for proficiency in DNA repair, recombination, and genome stability in Ustilago maydis

In a screen for DNA repair-defective mutants in the fungus Ustilago maydis, a gene encoding a BRCA2 family member, designated here as Brh2, was identified. A brh2 null allele was found to be defective in allelic recombination, meiosis, and repair of gaps and ionizing radiation damage to the same extent as rad51. Frequent marker loss in meiosis and diploid formation suggested that genomic instability was associated with brh2. This notion was confirmed by molecular karyotype analysis, which revealed gross chromosomal alterations associated with brh2. Yeast two-hybrid analysis indicated interaction between Brh2 and Rad51. Recapitulation in U. maydis of defects in DNA repair and genome stability associated with brh2 means that the BRCA2 gene family is more widespread than previously thought