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61. Pseudomonas putida AlkA and AlkB Proteins Comprise Different Defense Systems for the Repair of Alkylation Damage to DNA – In Vivo, In Vitro, and In Silico Studies

Author

Mielecki, Damian, primary, Saumaa, Signe, additional, Wrzesiński, Michał, additional, Maciejewska, Agnieszka M., additional, Żuchniewicz, Karolina, additional, Sikora, Anna, additional, Piwowarski, Jan, additional, Nieminuszczy, Jadwiga, additional, Kivisaar, Maia, additional, and Grzesiuk, Elżbieta, additional

Published
2013
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63. Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target

Author

Tavita, Kairi, primary, Mikkel, Katren, additional, Tark-Dame, Mariliis, additional, Jerabek, Hansjoerg, additional, Teras, Riho, additional, Sidorenko, Julia, additional, Tegova, Radi, additional, Tover, Andres, additional, Dame, Remus T., additional, and Kivisaar, Maia, additional

Published
2012
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65. Ongoing evolution of Pseudomonas aeruginosaPAO1 sublines complicates studies of DNA damage repair and tolerance

Author

Sidorenko, Julia, Jatsenko, Tatjana, and Kivisaar, Maia

Abstract

•Microevolution of P. aeruginosaPAO1 sublines leads to discordant phenotypes.•MPAO1 and its two-allele transposon mutant library cannot be used comparatively.•DNA damage inducible mutagenesis cassette is absent in some PAO1 sublines.•Functions of DNA damage repair and tolerance systems are masked in MPAO1 subline.

Published
2017
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72. Freeing P seudomonas putida KT2440 of its proviral load strengthens endurance to environmental stresses.

Author

Martínez ‐ García, Esteban, Jatsenko, Tatjana, Kivisaar, Maia, and Lorenzo, Víctor

Subjects
EFFECT of stress on bacteria, SOIL microbiology, BACTERIAL genomes, PSEUDOMONAS putida, LYSIS, PHENOTYPES, GENETIC recombination
Abstract

2.6% of the genome of the soil bacterium P seudomonas putida KT2440 encodes phage-related functions, but the burden of such opportunistic DNA on the host physiology is unknown. Each of the four apparently complete prophages borne by this strain was tested for stability, spontaneous excision and ability to cause lysis under various stressing conditions. While prophages P3 ( PP2266- PP2297) and P4 ( PP1532-1584) were discharged from the genome at a detectable rate, their induction failed otherwise to yield infective viruses. Isogenic P . putida KT2440 derivatives bearing single and multiple deletions of each of the prophages were then subjected to thorough phenotypic analyses, which generally associated the loss of proviral DNA with an increase of physiological vigour. The most conspicuous benefit acquired by prophage-less cells was a remarkable improvement in tolerance to UV light and other insults to DNA. This was not accompanied, however, with an upgrade of recA-mediated homologous recombination. The range of tolerance to DNA damage gained by the prophage-free strain was equivalent to the UV resistance endowed by the TOL plasmid pWW0 to the wild-type bacterium. While the P . putida's prophages are therefore genuinely parasitic, their detrimental effects can be offset by acquisition of compensatory traits through horizontal gene transfer. [ABSTRACT FROM AUTHOR]

Published
2015
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85. The ColR–ColS two-component signal transduction system is involved in regulation of Tn4652transposition inPseudomonas putidaunder starvation conditions.

Author

Hörak, Ria, Ilves, Heili, Pruunsild, Priit, Kuljus, Martin, and Kivisaar, Maia

Subjects
CELLULAR signal transduction, CHROMOSOMAL translocation, BACTERIA, GENETIC transduction, PHOSPHORESCENCE, MUTAGENESIS
Abstract

Bacteria use two-component signal transduction pathways to sense both extracellular and intracellular environment and to coordinate cellular events according to changing conditions. Adaptation can be either physiological or genetical. Here, we present evidence that a genome reorganization process such as transposition can be controlled by certain environmental cues sensed by a two-component signal transduction system. We demonstrate that transposition-dependent accumulation of phenol-utilizing mutants is severely decreased inPseudomonas putidadefective in a two-component systemcolRS. Translocation of Tn4652is decreased both incolR- andcolS-defective strains, indicating that signal transduction from a histidine kinase ColS to a response regulator ColR is necessary for the activation of Tn4652in bacteria starving on phenol. However, overexpression of ColR in acolS-defective strain restores Tn4652transposition, suggesting that absence of the signal from ColS can be compensated by an elevated amount of ColR.In vitroanalysis of purified ColR and ColS proteins evidenced that they constitute a functional phosphorelay. Site-directed mutagenesis revealed that a conserved H221 can be the phosphoryl-accepting residue in ColS and that aspartate residues D8 and D51 of ColR are necessary for the phosphotransfer from ColS to ColR. To our knowledge, Tn4652is the first bacterial transposon regulated by a two-component system. This finding indicates that transpositional activity can respond to signals sensed and processed by the host. [ABSTRACT FROM AUTHOR]

Published
2004
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86. IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn 4652 in stationary phase.

Author

Lives, Heili, Hörak, Rita, Teras, Riho, and Kivisaar, Maia

Subjects
MOBILE genetic elements, CHROMOSOMAL translocation, PSEUDOMONAS, TRANSPOSONS, DNA, MOLECULAR genetics
Abstract

Transpositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn 4652 in Pseudomonas putida is a stationary phase-specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445–5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida, might contribute to the transposition of Tn 4652 as well. Here, we demonstrate that transposition of Tn 4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn 4652 occurs in a P. putida ihfA-defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild-type strain. This indicates that the amount of IHF is a bottleneck in Tn 4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase. [ABSTRACT FROM AUTHOR]

Published
2004
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87. Homologous recombination is facilitated in starving populations of Pseudomonas putidaby phenol stress and affected by chromosomal location of the recombination target

Author

Tavita, Kairi, Mikkel, Katren, Tark-Dame, Mariliis, Jerabek, Hansjoerg, Teras, Riho, Sidorenko, Julia, Tegova, Radi, Tover, Andres, Dame, Remus T., and Kivisaar, Maia

Abstract

► Homologous recombination increases in starving P. putidain the presence of phenol. ► Reactive oxygen species stimulate homologous recombination. ► Emergence of recombinants is suppressed by DNA mismatch repair enzymes and RpoS. ► Chromosomal location of the target influences frequency of homologous recombination. ► In silico analysis of binding sites of NAPs at homologous recombination target sites.

Published
2012
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88. Pseudouridines of tRNA Anticodon Stem-Loop Have Unexpected Role in Mutagenesis in Pseudomonas sp.

Author

Tagel, Mari, Ilves, Heili, Leppik, Margus, Jürgenstein, Karl, Remme, Jaanus, and Kivisaar, Maia

Subjects
PSEUDOMONAS, MUTAGENESIS, DNA damage, PROTEOMICS, PSEUDOMONAS putida, DNA polymerases, TRANSFER RNA, PSEUDOMONAS aeruginosa
Abstract

Pseudouridines are known to be important for optimal translation. In this study we demonstrate an unexpected link between pseudouridylation of tRNA and mutation frequency in Pseudomonas species. We observed that the lack of pseudouridylation activity of pseudouridine synthases TruA or RluA elevates the mutation frequency in Pseudomonas putida 3 to 5-fold. The absence of TruA but not RluA elevates mutation frequency also in Pseudomonas aeruginosa. Based on the results of genetic studies and analysis of proteome data, the mutagenic effect of the pseudouridylation deficiency cannot be ascribed to the involvement of error-prone DNA polymerases or malfunctioning of DNA repair pathways. In addition, although the deficiency in TruA-dependent pseudouridylation made P. putida cells more sensitive to antimicrobial compounds that may cause oxidative stress and DNA damage, cultivation of bacteria in the presence of reactive oxygen species (ROS)-scavenging compounds did not eliminate the mutator phenotype. Thus, the elevated mutation frequency in the absence of tRNA pseudouridylation could be the result of a more specific response or, alternatively, of a cumulative effect of several small effects disturbing distinct cellular functions, which remain undetected when studied independently. This work suggests that pseudouridines link the translation machinery to mutation frequency. [ABSTRACT FROM AUTHOR]

Published
2021
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89. Mutation and Recombination Rates Vary Across Bacterial Chromosome.

Author

Kivisaar, Maia

Subjects
BACTERIAL chromosomes, NUCLEOIDS, RECOMBINANT DNA, BACTERIAL DNA, BACTERIAL evolution, CARRIER proteins, KARYOTYPES, HUMAN chromosomes
Abstract

Bacteria evolve as a result of mutations and acquisition of foreign DNA by recombination processes. A growing body of evidence suggests that mutation and recombination rates are not constant across the bacterial chromosome. Bacterial chromosomal DNA is organized into a compact nucleoid structure which is established by binding of the nucleoid-associated proteins (NAPs) and other proteins. This review gives an overview of recent findings indicating that the mutagenic and recombination processes in bacteria vary at different chromosomal positions. Involvement of NAPs and other possible mechanisms in these regional differences are discussed. Variations in mutation and recombination rates across the bacterial chromosome may have implications in the evolution of bacteria. [ABSTRACT FROM AUTHOR]

Published
2020
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91. Quorum sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: role in virulence and interspecies interactions in the olive knot

Author

Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, Kivisaar, Maia, Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, and Kivisaar, Maia

Abstract

The olive-knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi (PSV). PSV in the olive-knot undergoes interspecies interactions with the harmless endophyte Erwina toletana (ET); PSV and ET co-localize and form a stable community resulting in a more aggressive disease. PSV and ET produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal and they share AHLs in planta. In this work we have further studied the AHL QS systems of PSV and ET in order to determine possible molecular mechanism(s) involved in this bacterial inter-species interaction/cooperation. The AHL QS regulons of PSV and ET were determined allowing the identification of several QS-regulated genes. Surprisingly, the PSV QS regulon consisted of only a few loci whereas in ET many putative metabolic genes were regulated by QS among which several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which resulted to be essential for both co-localization of PSV and ET cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome. SIGNIFICANCE OF THIS STUDY: This is a report on studies of the quorum sensing (QS) systems of olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive-knot cooperator Erwinia toletana. These two bacterial species form a stable community in the olive knot, share QS signals and cooperate resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.

92. Quorum sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: role in virulence and interspecies interactions in the olive knot

Author

Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, Kivisaar, Maia, Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, and Kivisaar, Maia

Abstract

The olive-knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi (PSV). PSV in the olive-knot undergoes interspecies interactions with the harmless endophyte Erwina toletana (ET); PSV and ET co-localize and form a stable community resulting in a more aggressive disease. PSV and ET produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal and they share AHLs in planta. In this work we have further studied the AHL QS systems of PSV and ET in order to determine possible molecular mechanism(s) involved in this bacterial inter-species interaction/cooperation. The AHL QS regulons of PSV and ET were determined allowing the identification of several QS-regulated genes. Surprisingly, the PSV QS regulon consisted of only a few loci whereas in ET many putative metabolic genes were regulated by QS among which several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which resulted to be essential for both co-localization of PSV and ET cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome. SIGNIFICANCE OF THIS STUDY: This is a report on studies of the quorum sensing (QS) systems of olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive-knot cooperator Erwinia toletana. These two bacterial species form a stable community in the olive knot, share QS signals and cooperate resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.

93. Quorum sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: role in virulence and interspecies interactions in the olive knot

Author

Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, Kivisaar, Maia, Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, and Kivisaar, Maia

Abstract

The olive-knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi (PSV). PSV in the olive-knot undergoes interspecies interactions with the harmless endophyte Erwina toletana (ET); PSV and ET co-localize and form a stable community resulting in a more aggressive disease. PSV and ET produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal and they share AHLs in planta. In this work we have further studied the AHL QS systems of PSV and ET in order to determine possible molecular mechanism(s) involved in this bacterial inter-species interaction/cooperation. The AHL QS regulons of PSV and ET were determined allowing the identification of several QS-regulated genes. Surprisingly, the PSV QS regulon consisted of only a few loci whereas in ET many putative metabolic genes were regulated by QS among which several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which resulted to be essential for both co-localization of PSV and ET cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome. SIGNIFICANCE OF THIS STUDY: This is a report on studies of the quorum sensing (QS) systems of olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive-knot cooperator Erwinia toletana. These two bacterial species form a stable community in the olive knot, share QS signals and cooperate resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.

94. Quorum sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: role in virulence and interspecies interactions in the olive knot

Author

Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, Kivisaar, Maia, Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, and Kivisaar, Maia

Abstract

The olive-knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi (PSV). PSV in the olive-knot undergoes interspecies interactions with the harmless endophyte Erwina toletana (ET); PSV and ET co-localize and form a stable community resulting in a more aggressive disease. PSV and ET produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal and they share AHLs in planta. In this work we have further studied the AHL QS systems of PSV and ET in order to determine possible molecular mechanism(s) involved in this bacterial inter-species interaction/cooperation. The AHL QS regulons of PSV and ET were determined allowing the identification of several QS-regulated genes. Surprisingly, the PSV QS regulon consisted of only a few loci whereas in ET many putative metabolic genes were regulated by QS among which several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which resulted to be essential for both co-localization of PSV and ET cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome. SIGNIFICANCE OF THIS STUDY: This is a report on studies of the quorum sensing (QS) systems of olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive-knot cooperator Erwinia toletana. These two bacterial species form a stable community in the olive knot, share QS signals and cooperate resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.

95. Quorum sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: role in virulence and interspecies interactions in the olive knot

Author

Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, Kivisaar, Maia, Caballo-Ponce, Eloy, Meng, Xianfa, Uzelac, Gordana, Halliday, Nigel, Cámara, Miguel, Licastro, Danilo, Passos da Silva, Daniel, Ramos, Cayo, Venturi, Vittorio, and Kivisaar, Maia

Abstract

The olive-knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi (PSV). PSV in the olive-knot undergoes interspecies interactions with the harmless endophyte Erwina toletana (ET); PSV and ET co-localize and form a stable community resulting in a more aggressive disease. PSV and ET produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal and they share AHLs in planta. In this work we have further studied the AHL QS systems of PSV and ET in order to determine possible molecular mechanism(s) involved in this bacterial inter-species interaction/cooperation. The AHL QS regulons of PSV and ET were determined allowing the identification of several QS-regulated genes. Surprisingly, the PSV QS regulon consisted of only a few loci whereas in ET many putative metabolic genes were regulated by QS among which several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which resulted to be essential for both co-localization of PSV and ET cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome. SIGNIFICANCE OF THIS STUDY: This is a report on studies of the quorum sensing (QS) systems of olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive-knot cooperator Erwinia toletana. These two bacterial species form a stable community in the olive knot, share QS signals and cooperate resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.

96. Pseudomonas putida AlkA and AlkB Proteins Comprise Different Defense Systems for the Repair of Alkylation Damage to DNA – In Vivo, In Vitro, and In Silico Studies.

Author

Mielecki, Damian, Saumaa, Signe, Wrzesiński, Michał, Maciejewska, Agnieszka M., Żuchniewicz, Karolina, Sikora, Anna, Piwowarski, Jan, Nieminuszczy, Jadwiga, Kivisaar, Maia, and Grzesiuk, Elżbieta

Subjects
PSEUDOMONAS putida, DNA alkylation, DNA damage, ENVIRONMENTAL chemistry, DNA repair, BIOLOGICAL adaptation, GENE expression
Abstract

Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo- and endogenous origin. [ABSTRACT FROM AUTHOR]

Published
2013
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97. Involvement of sigma[sup S]in Starvation-Induced Transposition of Pseudomonas putida Transposon....

Author

Ilves, Heili, Horak, Rita, and Kivisaar, Maia

Subjects
*SIGMA particles, *BACTERIAL genetics, *PSEUDOMONAS
Abstract

Examines the involvement of sigma[sup S] in starvation-induced transposition of Pseudomonas putida transposon Tn4652. Induction of transpositional activity by different environmental stresses; Evidence on the transposition of Tn4652; Control of transcription from the Tn4652 by the stationary-phase specific sigma factor.

Published
2001
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98. Mutatsioonisagedust mõjutavate tegurite otsinguil: tRNA modifikatsiooniensüümid TruA ja RluA mutatsiooniprotsessides

Author

Tagel, Mari, Kivisaar, Maia, juhendaja, Ilves, Heili, juhendaja, Remme, Jaanus, juhendaja, and Tartu Ülikool. Loodus- ja täppisteaduste valdkond

Subjects
dissertations, ensüümid, Pseudomonas putida, mutagenicity tests, dissertatsioonid, enzymes, modifications, ETD, mutations, väitekirjad, bakterid, geenid, modifikatsioonid, transport RNA, bacteria, genes, mutageensustestid, tRNA, mutatsioonid
Abstract

Väitekirja elektrooniline versioon ei sisalda publikatsioone, Bakterid suudavad elada kõikjal, kuid karmide ja muutlike keskkonnatingimustega kohanemiseks on aga vaja geneetilist varieeruvust. Bakterites on selle põhiliseks allikaks mutatsioonid. Evolutsiooni mõistmiseks on vaja selgitada molekulaarseid mehhanisme, mis mõjutavad mutatsioonide tekkesagedust. Käesolevas töös kirjeldasin ja analüüsisin uut testsüsteemi, mis võimaldab tuvastada mutatsioonisagedust mõjutavaid faktoreid bakteriperekonnas Pseudomonas. Kirjeldatud testsüsteemi abil õnnestus mullabakteris Pseudomonas putida tuvastada nii varem kirjeldatud kui ka uusi mutatsioonisagedust mõjutavaid geene. Üllatavaim leid oli tRNA modifikatsiooniensüümide TruA ja RluA mõju mutatsioonisagedusele. tRNAd on väikesed molekulid, mis valgusünteesil kannavad valkude ehituskive ribosoomi. Selleks, et paremini oma funktsiooni täita, on paljud nukleotiidid tRNAdes modifitseeritud. Modifikatsioonidel võib olla palju ülesandeid, näiteks aitavad modifikatsioonid tRNAdel saavutada õiget struktuuri või suurendavad translatsiooni täpsust. TruA ja RluA modifitseerivad U nukleotiidi pseudouridiiniks, tehes seda erineval poolel tRNA antikoodonist. Näitasime, et TruA ja RluA tehtud modifikatsioonide puudumisel suureneb P. putidas mutatsioonisagedus. Mõistmaks paremini nende ensüümide olulisust, analüüsisime translatsiooni täpsust, stressi taluvust, proteoomi ja üldist elulemust P. putida TruA ja RluA defektsetes tüvedes. Lisaks sellele selgitasime võrdlevalt TruA ja RluA rolle ka Pseudomonas aeruginosa ja Escherichia coli rakkudes. Saadud tulemustest on näha, kuidas konserveerunud funktsiooniga valgud võivad põhjustada erinevates bakterites erinevaid fenotüüpe. Samuti ilmestab käesolev töö, kuivõrd mitmekesised ja ootamatud tegurid võivad mõjutada DNAs mutatsioonide teket., Bacteria can live everywhere. To cope with harsh and everchanging environmental conditions there is a constant need for genetic versatility. Mutations are the main source of genetic versatility in bacteria. To understand the evolution and adaptive abilities of bacteria, it is vital to investigate the processes affecting the mutation frequency. We have created, verified, and applied a new test system for detecting factors affecting the mutation frequency in bacteria from the genus Pseudomonas. By exploiting the new assay, we identified several genes affecting the mutation frequency in the soil bacterium Pseudomonas putida, many of which were not previously associated with the mutation frequency. Most surprising finding was that the tRNA modification enzymes TruA and RluA affect mutagenesis. tRNAs are small adaptor molecules that carry the building blocks of enzymes to the ribosome and thus are essential for translation. To improve their performance, tRNAs are extensively modified. Among other roles, modifications help tRNA’s to achieve the correct structure and improve the translation fidelity. TruA and RluA modify U nucleotide into pseudouridines in the close vicinity of tRNA anticodon. We demonstrated that the lack of TruA- and RluA-catalyzed modifications remarkably increases the mutation frequency in P. putida. To further analyze the importance of the modification enzymes TruA and RluA, we measured the stress tolerance, translation fidelity, protein expression and general fitness of P. putida strains lacking TruA or RluA. For comparison we analyzed the phenotypes caused by TruA and RluA deficiency in Pseudomonas aeruginosa and Escherichia coli cells. Our research demonstrates how an enzyme with conserved function can cause diverse phenotypes in different bacteria. Also, the thesis illustrates how the complex world of DNA mutations can be affected by many nonobvious factors.

Published
2022

99. Retracted and Republished from: 'Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola'

Author

Benjamin W. Held, Robert A. Blanchette, Jonathan S. Schilling, Anna Lipzen, Steven R. Ahrendt, Baojun Wu, Igor V. Grigoriev, Cristina Toapanta, Emma R. Master, Dan Cullen, David S. Hibbett, Jiwei Zhang, Thu V. Vuong, Jill Gaskell, and Kivisaar, Maia

Subjects
Pinicola, Fungus, Fomitopsis pinicola, Applied Microbiology and Biotechnology, complex mixtures, Microbiology, decay, 03 medical and health sciences, lignocellulose, Polypore, Botany, Environmental Microbiology, Genetics, Aryl-alcohol oxidase, Polyporales, 030304 developmental biology, Laccase, 0303 health sciences, Ecology, biology, 030306 microbiology, Host (biology), technology, industry, and agriculture, basidiomycetes, brown rot, biology.organism_classification, transcriptome, Food Science, Biotechnology
Abstract

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species—aspen, pine, and spruce—under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Published
2021

100. Repeated Exposure of Aspergillus niger Spores to the Antifungal Bacterium Collimonas fungivorans Ter331 Selects for Delayed Spore Germination

Author

Ioannis Stergiopoulos, Johannes Henricus Josephus Leveau, Sandra Mosquera, and Kivisaar, Maia

Subjects
Spores, Fungus, Applied Microbiology and Biotechnology, Microbiology, Microbial Ecology, 03 medical and health sciences, Rare Diseases, Oxalobacteraceae, spore inhibition, Spore germination, 2.2 Factors relating to the physical environment, germination asynchrony, Aetiology, Collimonas fungivorans, Mycelium, 030304 developmental biology, 0303 health sciences, Experimental evolution, Ecology, biology, 030306 microbiology, spore dormancy, Spores, Fungal, biology.organism_classification, Spore, spore germination, Fungal, Infectious Diseases, Emerging Infectious Diseases, Germination, Microbial Interactions, antagonistic bacterial-fungal interaction, Aspergillus niger, Infection, Bacteria, Food Science, Biotechnology
Abstract

The bacterial strain Collimonas fungivorans Ter331 (CfTer331) inhibits mycelial growth and spore germination in Aspergillus niger N402 (AnN402). The mechanisms underlying this antagonistic bacterial-fungal interaction have been extensively studied, but knowledge on the long-term outcome of this interaction is currently lacking. Here, we used experimental evolution to explore the dynamics of fungal adaptation to recurrent exposure to CfTer331. Specifically, five single-spore isolates (SSIs) of AnN402 were evolved under three selection scenarios in liquid culture, i.e., (i) in the presence of CfTer331 for 80 growth cycles, (ii) in the absence of the bacterium for 80 cycles, and (iii) in the presence of CfTer331 for 40 cycles and then in its absence for 40 cycles. The evolved SSI lineages were then evaluated for phenotypic changes from the founder fungal strain, such as germinability with or without CfTer331. The analysis showed that recurrent exposure to CfTer331 selected for fungal lineages with reduced germinability and slower germination, even in the absence of CfTer331. In contrast, when AnN402 evolved in the absence of the bacteria, lineages with increased germinability and faster germination were favored. SSIs that were first evolved in the presence of CfTer331 and then in its absence showed intermediate phenotypes but overall were more similar to SSIs that evolved in the absence of CfTer331 for 80 cycles. This suggests that traits acquired from exposure to CfTer331 were reversible upon removal of the selection pressure. Overall, our study provides insights into the effects on fungi from the long-term coculture with bacteria. IMPORTANCE The use of antagonistic bacteria for managing fungal diseases is becoming increasingly popular, and thus there is a need to understand the implications of their long-term use against fungi. Most efforts have so far focused on characterizing the antifungal properties and mode of action of the bacterial antagonists, but the possible outcomes of the persisting interaction between antagonistic bacteria and fungi are not well understood. In this study, we used experimental evolution in order to explore the evolutionary aspects of an antagonistic bacterial-fungal interaction, using the antifungal bacterium Collimonas fungivorans and the fungus Aspergillus niger as a model system. We show that evolution in the presence or absence of the bacteria selects for fungal lineages with opposing and conditionally beneficial traits, such as slow and fast spore germination, respectively. Overall, our studies reveal that fungal responses to biotic factors related to antagonism could be to some extent predictable and reversible.

Published
2021

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