Search

DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli. We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals., In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous, and important intermediate for various chemicals in industry, by analyzing its metabolism at the system level in Escherichia coli. Using constraint-based modeling, we show that the growth of E. coli on DHA is suboptimal and identify the potential causes. Nuclear magnetic resonance analysis shows that DHA is degraded nonenzymatically into substrates known to be unfavorable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e., the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobically mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA. IMPORTANCE DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli. We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals.

The olive knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi. P. savastanoi pv. savastanoi in the olive knot undergoes interspecies interactions with the harmless endophyte Erwinia toletana; P. savastanoi pv. savastanoi and E. toletana colocalize and form a stable community, resulting in a more aggressive disease. P. savastanoi pv. savastanoi and E. toletana 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 P. savastanoi pv. savastanoi and E. toletana in order to determine possible molecular mechanism(s) involved in this bacterial interspecies interaction/cooperation. The AHL QS regulons of P. savastanoi pv. savastanoi and E. toletana were determined, allowing the identification of several QS-regulated genes. Surprisingly, the P. savastanoi pv. savastanoi QS regulon consisted of only a few loci whereas in E. toletana many putative metabolic genes were regulated by QS, among which are several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which was found to be essential for both colocalization of P. savastanoi pv. savastanoi and E. toletana cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome. IMPORTANCE This is a report on studies of the quorum sensing (QS) systems of the 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 as by 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.

2.6% of the genome of the soil bacterium Pseudomonas 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., This study was supported by the BIO Program of the Spanish Ministry of Science and Innovation, the ST-FLOW and ARYSIS Contracts of the EU, the ERANET-IB Program and the PROMT Project of the CAM. The work in MK Laboratory is supported by Estonian Science Foundation, grant number 9114 to MK, by Estonian Ministry of Research Targeted Financing Project SF0180031s08.

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.

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.

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.

Väitkirja elektrooniline versioon ei sisalda publikatsioone, Enamuse oma elust looduslikes tingimustes on bakterid toitainete näljas ja stressis, mis tuleneb pidevalt muutuvatest keskkonnatingimustest. Üheks mooduseks uute tingimustega kohaneda on mutatsioonide teke, mis võimaldab bakterite kiiret evolutsioneerumist. Kuna mutatsioonid võimaldavad bakteritel saavutada resistentsuse antibiootikumide suhtes või omandada kaitse peremeesorganismis immuunsüsteemi rünnaku eest, on oluline mõista mehhanisme, mis soodustavad geneetiliste muutuste teket. On välja pakutud, et eluks hädavajalik protsess RNA süntees ehk transkriptsioon võib soodustada mutatsioonide teket, kuid transkriptsiooni täpne roll ja mehhanism mutatsiooniprotsessides on veel selgitamisel. Lisaks on ebaselge transkriptsiooniga kaasneva DNA repartasiooni roll mutatsioonide tekkes. Bakterites vahendavad transkriptsioonseoselist reparatsiooni valgud Mfd ja UvrD. Kuigi transkriptsiooniseoseline reparatsioon eemaldab DNA-lt vigu ja peaks seetõttu vähendama mutatsioonide tekkesagedust, on mitmed uurimustööd hiljuti näidanud, et Mfd-st sõltuv rada osaleb ka mutatsioonide tekitamises. Antud doktoritöö eesmärk oli välja selgitada transkriptsiooni ja transkriptsiooniseoselist reparatsiooni vahendavate valkude Mfd ja UvrD mõju mutatsioonide tekkele mullabakteris Pseudomonas putida. See bakter kuulub suurde pseudomonaadide perekonda, mille liikmed on metaboolselt väga mitmekülgsed ja võimelised kiiresti kohanema muutuvate keskkonnatingimustega. Mitmed selle perekonna liikmed on huvi pakkuvad ka oma biotehnoloogilise rakendatavuse või patogeensuse tõttu. Doktoritöö tulemused näitavad, et tranksriptsioon on rakusisene protsess, mis soodustab geneetiliste muutuste teket bakteris P. putida - testgeeni transkriptsiooni taseme tõstmisel suurenes ka mutatsioonide tekkesagedust. Ka transkriptsiooniga kaasneval DNA parandamisel on roll mutatsioonilistes protsessides - UvrD valk osaleb mutatsioonide ära hoidmises, samas kui Mfd soodustab teatud tingimustes mutatsioonide teket ja võib seeläbi kaasa aidata bakterite evolutsioneerumisele, For most of their lives in nature bacteria are nutrient-deprived and stressed due to ever-changing environmental conditions. To survive, microbial populations can rapidly evolve through formation of mutations. As mutations enable the bacteria to develop resistance to antibiotics and acquire protection to hosts immune response, it is necessary to understand the mechanisms that promote mutagenic processes. It has been proposed that the essential process of RNA synthesis, i.e. transcription, is also a factor promoting formation of mutations. However, the exact mechanism and role of transcription in mutagenic processes is still unknown. In addition, it is unclear how the DNA repair coupled with transcription affects the occurrence of mutations. In bacteria transcription-coupled repair is mediated by repair proteins Mfd and UvrD. While transcription-coupled repair removes DNA lesions and should thereby maintain the genome integrity, accumulating body of evidence suggesting that in some circumstances transcription-coupled repair may instead induce the occurrence of mutations through the Mfd-dependent pathway. In this thesis the role of transcription and transcription-coupled repair factors Mfd and UvrD in mutational processes was studied in soil bacterium Pseudomonas putida. This bacterium is a member of a large Pseudomonas genus, which comprises a diverse group of bacteria that are metabolically diverse and have a broad potential for adaptation to changing environmental conditions. Several members are also of great interest because of their prospects in biotechnological applications or their ability to cause disease in plants and animals. Results of the thesis demonstrate that transcription is an endogenous process that promotes occurrence of genetic changes in P. putida - elevated transcription of the mutational target gene results in elevated mutation frequency. Repair coupled with transcription also has a role in the mutagenic processes - UvrD is essential for preventing occurrence of mutations, while Mfd promotes the occurrence of mutations in some conditions and may thus contribute to the evolution of bacteria, https://www.ester.ee/record=b5359561

Antiseptics and disinfectant products are of great importance to control and eliminate pathogens, especially in settings such as hospitals and the food industry. Such products are widely distributed and frequently poorly regulated. Occasional outbreaks have been associated with microbes resistant to such compounds, and researchers have indicated potential cross-resistance with antibiotics. Despite that, there are many gaps in knowledge about the bacterial stress response and the mechanisms of microbial resistance to antiseptics and disinfectants. We investigated the stress response of the bacterium Escherichia coli to 10 common disinfectant and antiseptic chemicals to shed light on the potential mechanisms of tolerance to such compounds., The mechanisms of the bacterial response to biocides are poorly understood, despite their broad application. To identify the genetic basis and pathways implicated in the biocide stress response, we exposed Escherichia coli populations to 10 ubiquitous biocides. By comparing the transcriptional responses between a short-term exposure (30 min) and a long-term exposure (8 to 12 h) to biocide stress, we established the common gene and pathway clusters that are implicated in general and biocide-specific stress responses. Our analysis revealed a temporal choreography, starting from the upregulation of chaperones to the subsequent repression of motility and chemotaxis pathways and the induction of an anaerobic pool of enzymes and biofilm regulators. A systematic analysis of the transcriptional data identified a zur-regulated gene cluster to be highly active in the stress response against sodium hypochlorite and peracetic acid, presenting a link between the biocide stress response and zinc homeostasis. Susceptibility assays with knockout mutants further validated our findings and provide clear targets for downstream investigation of the implicated mechanisms of action. IMPORTANCE Antiseptics and disinfectant products are of great importance to control and eliminate pathogens, especially in settings such as hospitals and the food industry. Such products are widely distributed and frequently poorly regulated. Occasional outbreaks have been associated with microbes resistant to such compounds, and researchers have indicated potential cross-resistance with antibiotics. Despite that, there are many gaps in knowledge about the bacterial stress response and the mechanisms of microbial resistance to antiseptics and disinfectants. We investigated the stress response of the bacterium Escherichia coli to 10 common disinfectant and antiseptic chemicals to shed light on the potential mechanisms of tolerance to such compounds.

GacA/GacS two-component system can be found in Gram-negative bacteria, including enteric bacteria and Pseudomonas. gacS gene encodes for a membrane-bound sensor kinase GacS, whereas a transcriptional response regulator GacA is encoded by gacA gene. The aim of this thesis was to investigate whether the inactivation of the GacA/GacS two-component system could affect mutation frequency in Pseudomonas putida. Two test systems were employed for measuring mutation frequency: chromosomal RifR assay and a plasmidial test system based on lactose degradation. RifR phenotype of bacteria is a result of mutations that decrease the affinity of rifampicin binding to the β subunit of RNA polymerase. This makes this enzyme insensitive to rifampicin. The second test system is based on the monitoring mutations in lacZ gene encoding for β-galactosidase, which turns the tester strains from Lac- to Lac+ phenotype. Usage of both test systems revealed that the inactivation of the gacA gene elevates mutation frequency in P. putida. In estonian: GacA/GacS kahekomponendiline süsteem on kirjeldatud erinevates Gram negatiivsetes bakteriliikides, kuhu kuuluvad ka enterobakterid ja erinevad Pseudomonase liigid. Geen gacS kodeerib membraan-seoselist sensorkinaasi GacS ning transkriptsiooni regulaator GacA, mis saab signaali GacS-lt, on kodeeritud geeni gacA poolt. Käesoleva bakalaureusetöö eesmärgiks oli välja selgitada, kas GacA/GacS süsteemi inaktiveerimine mõjutab bakteris Pseudomonas putida mutatsioonisagedust. Mutatsioonisageduse mõõtmiseks kasutati kahte testsüsteemi: kromosomaalset RifR süsteemi ja plasmiidset laktoosi lagundamisel põhinevat Lac+ süsteemi. RifR fenotüübiga bakterites on tekkinud mutatsioonid, mis vähendavad rifampitsiini seondumist RNA polümeraasi β-subühikuga, muutes sel viisil ensüümi rifampitsiini suhtes tundetuks. Teine testsüsteem põhineb β-galaktosidaasi kodeerivas geenis lacZ tekkivate mutatsioonide tuvastamisel, mis võimaldavad Lac- testertüvedel hakata lagundama laktoosi (Lac+ reversioon). Töö tulemustena selgus, et gacA geeni inaktiveerimine bakteris P. putida põhjustas mutatsioonisageduse suurenemist mõlemate testsüsteemide rakendamisel.

Microbial food safety of raw or minimally processed fresh produce is a significant challenge. The current sanitation processes are effective for inactivation of bacteria in wash water but have limited efficacy (5-log reduction) in the presence of relatively high organic content (chemical oxygen demand of 500 mg/liter), demonstrating a potential to address a significant unmet need to improve fresh produce sanitation. The particle-based sanitizer had no significant effect on the quality of fresh lettuce.IMPORTANCE The limitation of current sanitation processes for inactivation of microbes on the surfaces of fresh produce is due to nonspecific consumption of sanitizers by reactions with the food matrix and complexity of surface chemistries and structural features of produce surfaces. This study demonstrates a novel approach to enhance sanitation effectiveness of fresh produce using a particle-based sanitizer. The particle-based sanitizer concept provides localized high concentration delivery of chlorine to the surfaces of fresh produce and enables more than 5 logs of inactivation of inoculated bacteria on fresh produce surfaces without significant changes in produce quality. The results of this study illustrate the potential of this approach to address the unmet need for improving sanitation of fresh produce. Further validation of this approach using a scaled-up produce washing system will enable commercialization of this novel concept.

Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria. Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria. This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB., Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacteria of the genus Magnetospirillum that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria. IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria. Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria. This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.

Väitekirja elektrooniline versioon ei sisalda publikatsioone, Kahjustused DNA-s, mis tekivad kas rakkude normaalse elutegevuse käigus või erinevate keskonnategurite mõjul (näiteks UV-kiirgus, DNA-d kahjustavad kemikaalid), pärsivad genoomi replikatsiooni, takistades replikatiivse DNA polümeraasi edasiliikumist. Pikaajaline replikatsiooni seiskumine võib osutuda rakkudele letaalseks. Selleks, et DNA replikatsioon saaks jätkuda ka kahjustatud DNA-lt, on välja kujunenud DNA kahjustuste tolereerimise mehhanismid. Üheks neist on DNA kahjustustest ülesüntees (translesion DNA synthesis, TLS), mida viivad läbi spetsialiseeritud DNA polümeraasid. Need polümeraasid jätkavad DNA sünteesi kahjustatud nukleotiidi kohalt, tagades organismi ellujäämise DNA kahjustuste olemasolul. Samas võib vigaderohke süntees viia mutatsioonide tekkeni, mis on alusmaterjaliks evolutsioonile, kuid põhjustavad ka geneetilisi haigusi. Näiteks bakteritel on TLS polümeraaside toimel tekkinud geneetiline variantsus oluline antibiootikumide resistentsuse ja infektsioonivõime kujunemisel. TLS polümeraasid on potentsiaalseks märklauaks nii antibakteriaalses ravis kui ka vähiteraapias. Minu doktoritöö eesmärgiks oli selgitada TLS polümeraaside funktsioone, eeskätt nende võimalikku rolli mutatsiooniprotsessides ning DNA kahjustuste tolereerimisel perekonda Pseudomonas kuuluval mullabakteril P. putida ja inimese oportunistlikul patogeenil P. aeruginosa. Pseudomonaadidel on kolm TLS polümeraasi: Pol II, Pol IV ja ImuABC. Uurimistöö tulemused viitavad sellele, et bakteris P. putida võivad TLS polümeraasid osaleda DNA sünteesil DNA polümeraasi Pol I puudumisel. Lisaks selgus, et Pol IV ja ImuABC on olulised DNA alküülkahjustuste talumisel. Kui ImuC viib läbi vigaderohket sünteesi, suurendades mutatsioonide arvu, siis Pol IV ületab DNA alküülkahjustusi väga täpselt. Üllatuslikult selgus, et bakterite inkubeerimise temperatuur mõjutab DNA alküülkahjustuste tolereerimist ja/või reparatsiooni efektiivsust., The integrity of our hereditary material is constantly challenged by both endogenous agents formed during normal cellular metabolism and by various exogenous factors, like UV-light and chemicals found everywhere in the environment. Damage in DNA can block genome replication, which can lead to genomic instability and death of the cell. To overcome these blocks, organisms have evolved DNA damage tolerance mechanisms that allow completion of DNA replication in the presence of damage. One of them is translesion DNA synthesis (TLS), which is mediated by specialized TLS DNA polymerases that are able replicate over the replication-blocking DNA damage. Information encoded by a damaged nucleotide is usually inaccurate, therefore TLS is inherently error-prone process. As such, TLS polymerases not only protect cells against DNA damage, but also represent a potential source of mutations – a material for evolution, but also a cause of disease in humans. In bacteria, genetic diversity generated by TLS polymerases is critical for the acquisition of antibiotic resistance. Therefore, a lot of research is now undertaken to use TLS polymerases as a target for cancer or antimicrobial treatment. In the present work, I describe the role of TLS polymerases in two representatives of Pseudomonas, important human pathogen Pseudomonas aeruginosa and soil bacterium Pseudomonas putida. These organisms possess three TLS polymerases: Pol II, Pol IV and ImuABC. The results of our work revealed that in P. putida TLS polymerases might leave mutagenic fingerprints in the genome during normal growth of bacteria, suggesting their potential role in bypass past endogenously formed DNA damage. Moreover, Pol II and Pol IV might be involved in DNA replication in the absence of replicative DNA polymerase I. Both Pol IV and ImuABC in Pseudomonads are involved in DNA alkylation damage tolerance. Replication across alkylation damage by ImuABC is highly mutagenic, Pol IV, on the contrary, performs very accurate bypass. One of the important findings of my study was that simple switch in growth temperature of bacteria changed the cell’s strategy to deal with alkylation DNA damage and role of TLS in it.

A mechanistic understanding of how new phenotypes develop to overcome the loss of a gene product provides valuable insight on both the metabolic and regulatory function of the lost gene. Thepgigene, whose product catalyzes the second step in glycolysis, was deleted in a growth optimizedEscherichia coliK-12 MG1655 strain. The knock-out (KO) strain exhibited an 80% drop in growth rate, that was largely recovered in eight replicate, but phenotypically distinct, cultures after undergoing adaptive laboratory evolution (ALE). Multi omic data sets showed that the loss ofpgisubstantially shifted pathway usage leading to a redox and sugar phosphate stress response. These stress responses were overcome by unique combinations of innovative mutations selected for by ALE. Thus, we show the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after loss of a major gene product.ImportanceA mechanistic understanding of how new phenotypes develop to overcome the loss of a gene product provides valuable insight on both the metabolic and regulatory function of the lost gene. Thepgigene, whose product catalyzes the second step in glycolysis, was deleted in a growth optimizedEscherichia coliK-12 MG1655 strain. Eight replicate adaptive laboratory evolution (ALE) resulted in eight phenotypically distinct endpoints that were able to overcome the gene loss. Utilizing multi-omics analysis, we show the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after loss of a major gene product.

Adaptive laboratory evolution (ALE) experiments are often designed to maintain a static culturing environment to minimize confounding variables that could influence the adaptive process, but dynamic nutrient conditions occur frequently in natural and bioprocessing settings. To study the nature of carbon substrate fitness tradeoffs, we evolved batch cultures of Escherichia coli via serial propagation into tubes alternating between glucose and either xylose, glycerol, or acetate. Genome sequencing of evolved cultures revealed several genetic changes preferentially selected for under dynamic conditions and different adaptation strategies depending on the substrates being switched between; in some environments, a persistent “generalist” strain developed, while in another, two “specialist” subpopulations arose that alternated dominance. Diauxic lag phenotype varied across the generalists and specialists, in one case being completely abolished, while gene expression data distinguished the transcriptional strategies implemented by strains in pursuit of growth optimality. Genome-scale metabolic modeling techniques were then used to help explain the inherent substrate differences giving rise to the observed distinct adaptive strategies. This study gives insight into the population dynamics of adaptation in an alternating environment and into the underlying metabolic and genetic mechanisms. Furthermore, ALE-generated optimized strains have phenotypes with potential industrial bioprocessing applications. IMPORTANCE Evolution and natural selection inexorably lead to an organism's improved fitness in a given environment, whether in a laboratory or natural setting. However, despite the frequent natural occurrence of complex and dynamic growth environments, laboratory evolution experiments typically maintain simple, static culturing environments so as to reduce selection pressure complexity. In this study, we investigated the adaptive strategies underlying evolution to fluctuating environments by evolving Escherichia coli to conditions of frequently switching growth substrate. Characterization of evolved strains via a number of different data types revealed the various genetic and phenotypic changes implemented in pursuit of growth optimality and how these differed across the different growth substrates and switching protocols. This work not only helps to establish general principles of adaptation to complex environments but also suggests strategies for experimental design to achieve desired evolutionary outcomes.

The occurrence of mutations is a cornerstone of the evolutionary theory of adaptation, capitalizing on the rare chance that a mutation confers a fitness benefit. Natural selection is increasingly being leveraged in laboratory settings for industrial and basic science applications. Despite increasing deployment, there are no standardized procedures available for designing and performing adaptive laboratory evolution (ALE) experiments. Thus, there is a need to optimize the experimental design, specifically for determining when to consider an experiment complete and for balancing outcomes with available resources (i.e., laboratory supplies, personnel, and time). To design and to better understand ALE experiments, a simulator, ALEsim, was developed, validated, and applied to the optimization of ALE experiments. The effects of various passage sizes were experimentally determined and subsequently evaluated with ALEsim, to explain differences in experimental outcomes. Furthermore, a beneficial mutation rate of 10 −6.9 to 10 −8.4 mutations per cell division was derived. A retrospective analysis of ALE experiments revealed that passage sizes typically employed in serial passage batch culture ALE experiments led to inefficient production and fixation of beneficial mutations. ALEsim and the results described here will aid in the design of ALE experiments to fit the exact needs of a project while taking into account the resources required and will lower the barriers to entry for this experimental technique. IMPORTANCE ALE is a widely used scientific technique to increase scientific understanding, as well as to create industrially relevant organisms. The manner in which ALE experiments are conducted is highly manual and uniform, with little optimization for efficiency. Such inefficiencies result in suboptimal experiments that can take multiple months to complete. With the availability of automation and computer simulations, we can now perform these experiments in an optimized fashion and can design experiments to generate greater fitness in an accelerated time frame, thereby pushing the limits of what adaptive laboratory evolution can achieve.

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important. Kaikkien eliöiden perimä koostuu geeneistä jotka ohjeistavat elintärkeitä toimintoja, sekä ns. toistuvista DNA-jaksoista joiden osuus perimästä vaihtelee, ollen bakteereilla n. 3%, ihmisellä 45% ja joillain kasveilla jopa yli 70%. Näitä toistojaksoja pidettiin aikoinaan turhana "roska DNA:na" koska niiden toiminnasta ei ollut tietoa. Liikkuvat geneettiset elementit, "hyppivät geenit" eli transposonit ovat osa tätä toistuvaa DNA:ta. Tänä päivänä tiedetään että näillä elementeillä on tärkeä merkitys perimän evoluutiossa, ja jopa geenien synnyssä. Esimerkiksi nisäkkäiden immuunipuolustuksen vasta-ainegeenien uudelleenjärjestelymekanismin ajatellaan kehittyneen transposonisysteemistä. Bakteereilla antibioottien vastustuskykyä koodaavat geenit siirtyvät usein solusta toiseen transposonien välityksellä. Myös eräät virukset kuten HIV-1 ja bakteerivirus Mu toimivat kuten transposonit. Huolimatta transposonien monimuotoisuudesta ne kaikki käyttävät hyvin samanlaista liikkumismekanismia, transpositiota. Transposonit kokoavat transpositiokoneiston jossa yksinkertaisimmillaan transposaasi proteiinit ovat sitoutuneet transposoni DNA:n päihin muodostaen DNA-proteiinikompleksin (ydinkoneiston). Tässä kompleksissa transposaasi katalysoi kemialliset reaktiot, DNA:n katkaisun ja uudelleen liittämisen, ja tämän koneiston avulla transposoni liikkuu paikasta toiseen. Se kuinka nämä koneistot kootaan ja kuinka ne molekyylitasolla toimivat määrittää monia ominaisuuksia transposonin elinkierrossa. Tässä työssä tutkittiin kolmen transposonin transpositiokoneistoja, tunnistettiin niiden molekulaariset (proteiini ja DNA) komponentit, sekä tutkittiin niiden ominaispiirteitä ja toiminnallisuutta. Ensimmäisessä osassa tutkimme ohran retrotransposonia, BARE-1:a, jonka elämänkiertoon oletettavasti kuuluu RNA välivaihe ja viruksen kaltaiset partikkelit (VLP:t), jotka muistuttavat rakenteeltaan ja toiminnaltaan viruksia ja toimivat retrotransposonin transpositiokoneistona. Etsimme ohran solu-uutteista BARE-1:n VLP-koneiston komponentteja sekä koottuja VLP-koneistoja. Osoitimme että kaikki proteiini ja DNA komponentit jotka tarvitaan toiminnalliseen VLP-koneistoon voidaan havaita puhdistetuista ohran solu-uutteista. Elektronimikroskopian avulla havaitsimme myös rakenteita jotka muistuttavat viruksen kaltaisia partikkeleja. Työmme tulokset viittaavat siihen että BARE-1 voi olla aktiivinen retrotransposoni, joka kokoaa VLP-koneiston ja käyttää sitä liikkumiseen ohran perimässä. Toinen ja kolmas osa tutkimuksesta käsittelee transpositiota hyväksikäyttäviä bakteeriviruksia. Bakteerivirus Mu voi infektoida useita bakteerilajeja ja liittyä transpositiolla bakteerin perimään pysyen siellä piilevänä lisääntymättä (provirus). Vaihtoehtoisesti se voi lisääntyä bakteerisolussa monistumalla transposition avulla, jolloin isäntäsolu kuolee ja satoja uusia viruksia vapautuu etsimään uutta isäntäsolua. Mu on toiminut transposonitutkimuksen mallina, ja sen transpositiokoneiston toiminta tunnetaan tarkkaan, sillä sen liikkumismekanismina on jo pitkään voitu tutkia tehokkaasti ja yksinkertaisesti koeputkiolosuhteissa. Toiminnallinen Mu:n transpositiokoneisto voidaan koota koeputkessa käyttämällä transposaasiproteiini MuA:ta ja Mu:n DNA:n päitä. Tämän ydinkoneiston toimintaa voidaan tutkia molekyylitasolla. Muita Mu:n kaltaisia transposoniviruksia on tutkittu hyvin vähän. H. influenzae bakteerin perimässä on Mu-viruksen kaltainen DNA jakso, jonka nimesimme Hin-Mu profaagiksi. Tässä työssä määritimme Hin-Mu:n ydinkoneiston proteiini ja DNA komponentit, tuotimme ja puhdistimme ko. komponentteja ja tutkimme niiden ominaispiirteitä vertaamalla niitä Mu:n vastaaviin komponentteihin, ja osoitimme Hin-Mu:n ydinkoneiston olevan toiminnallinen vastaavissa koeputkiolosuhteissa joissa Mu:n transpositiota tutkitaan. Hin-Mu on ensimmäinen Mu:n kaltainen profaagi jonka transpositiokoneisto on tunnistettu ja osoitettu toiminnalliseksi. Työmme myös osoitti, että Mu:n ja Hin-Mu:n transpositiokoneistot ovat rakenteeltaan hyvin samankaltaisia, mutta omaavat tiettyjä toiminnallisia erilaisuuksia. Osoitimme myös että Mu transpositiokoneiston MuA transposaasi on hyvin joustava ja pystyy hyväksikäyttämään myös Hin-Mu:n transpositiokoneiston DNA komponentteja. Mu:n koneiston joustavuutta tutkittiin lisää käyttämällä ns. silmukkamuotoisia Mu DNA:n päitä (substraatteja), sillä tiedetään että osa transposoneista irrottautuu isäntänsä perimästä muodostamalla välituotteena ns. DNA:n silmukkarakenteen (hairpin). Tutkimme Mu:n koneiston kykyä käyttää hyväkseen tällaisia silmukkamuotoisia DNA substraatteja. Tutkimuksemme osoitti, että Mu:n transpositiokoneisto on niin joustava että se kykenee ottamaan erilaisia silmukkarakenteisia DNA substraatteja koneistona osaksi, avaamaan suljetut DNA silmukat, ja liittämään ne uuteen kohde DNA:han. Työ osoitti uudenlaista joustavuutta Mu:n transpositiokoneistolta ja MuA proteiinilta DNA substraattiin sitoutumisessa ja sen prosessoinnissa. Havaittu transpositiokoneiston joustavuus voi olla sekä evolutiivisesti että mekanistisesti tärkeää.