Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON. / Doctor of Philosophy / Metabolic engineering is the use of genetic engineering to modify microorganisms in order to produce high yields of valuable commodity chemicals. The goal of this research is to develop new methods to improve genetic modification and selection of microbial cells. The specific objectives were to: (i) develop new tools for DNA assembly and manipulation, (ii) utilize small synthetic RNA to control protein expression level, (iii) use Raman spectroscopy to study phenotypic responses to environmental changes and (iv) enrich for microorganisms that detoxify dangerous toxins. First, a new technique for DNA assembly, named λ-PCR (lambda-PCR), was developed. This method allows the easy manipulation of plasmid DNA with high-efficiency and low-cost compared to traditional techniques. Second, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system in order to modulate (downregulate or overexpress) fluorescent protein expression. Next, Raman spectroscopy was used to assess phenotypic response of cyanobacterial cells to different environmental modifications (light settings, salts, sugar, etc…). Finally, genomic library was used to discover and characterize enzymes capable of degrading a mycotoxin.

Yes / A crucial step in the transition from outcrossing to self-fertilization is the loss of genetic self-incompatibility (SI). In the Brassicaceae, SI involves the interaction of female and male speci-ficity components, encoded by the genesSRKandSCRat the self-incompatibility locus (S-lo-cus). Theory predicts thatS-linked mutations, and especially dominant mutations inSCR, arelikely to contribute to loss of SI. However, few studies have investigated the contribution ofdominant mutations to loss of SI in wild plant species. Here, we investigate the genetic basis of loss of SI in the self-fertilizing crucifer speciesCapsella orientalis, by combining genetic mapping, long-read sequencing of completeS-hap-lotypes, gene expression analyses and controlled crosses. We show that loss of SI inC. orientalisoccurred<2.6 Mya and maps as a dominant trait totheS-locus. We identify a fixed frameshift deletion in the male specificity geneSCRand con-firm loss of male SI specificity. We further identify anS-linked small RNA that is predicted tocause dominance of self-compatibility. Our results agree with predictions on the contribution of dominantS-linked mutations toloss of SI, and thus provide new insights into the molecular basis of mating system transitions. / Work at Uppsala Genome Center is funded by 550 RFI / VR and Science for Life Laboratory, Sweden. The SNP&SEQ Platform is supported by 551 the Swedish Research Council and the Knut and Alice Wallenberg Foundation. V.C. 552 acknowledges support by a grant from the European Research Council (NOVEL project, 553 grant #648321). The authors thank the French Ministère de l’Enseignement Supérieur et de la 554 Recherche, the Hauts de France Region and the European Funds for Regional Economical 555 Development for their financial support to this project. This work was supported by a grant 556 from the Swedish Research Council (grant #D0432001) and by a grant from the Science for 557 Life Laboratory, Swedish Biodiversity Program to T.S. The Swedish Biodiversity Program is 558 supported by the Knut and Alice Wallenberg Foundation.

La régulation de l’homéostasie du fer est cruciale chez les bactéries. Chez Salmonella, l’expression des gènes d’acquisition et du métabolisme du fer au moment approprié est importante pour sa survie et sa virulence. Cette régulation est effectuée par la protéine Fur et les petits ARN non codants RfrA et RfrB. Le rôle de ces régulateurs est d’assurer que le niveau de fer soit assez élevé pour la survie et le métabolisme de Salmonella, et assez faible pour éviter l’effet toxique du fer en présence d’oxygène. Les connaissances concernant le rôle de ces régulateurs ont été principalement obtenues par des études chez S. Typhimurium, un sérovar généraliste causant une gastro-entérite chez les humains. Très peu d’informations sont connues sur le rôle de ces régulateurs chez S. Typhi, un sérovar humain-spécifique responsable de la fièvre typhoïde. Le but de cette étude était de déterminer les rôles de Fur, RfrA et RfrB dans l’homéostasie du fer et la virulence de Salmonella, et de démontrer qu’ils ont une implication distincte chez les sérovars Typhi et Typhimurium. Premièrement, Fur, RfrA et RfrB régulent l’homéostasie du fer de Salmonella. Les résultats de cette étude ont démontré que Fur est requis pour la résistance au stress oxydatif et pour une croissance optimale dans différentes conditions in vitro. La sensibilité du mutant fur est due à l’expression des petits ARN RfrA et RfrB, et cette sensibilité est beaucoup plus importante chez S. Typhi que chez S. Typhimurium. Également, Fur inhibe la transcription des gènes codant pour les sidérophores en conditions riches en fer, tandis que les petits ARN RfrA et RfrB semblent être importants pour la production d’entérobactine et de salmochélines chez S. Typhi lors de conditions pauvres en fer. Ensuite, ces régulateurs affectent la virulence de Salmonella. Fur est important pour la motilité de Salmonella, particulièrement chez S. Typhi. Fur est nécessaire pour l’invasion des deux sérovars dans les cellules épithéliales, et pour l’entrée et la survie de S. Typhi dans les macrophages. Chez S. Typhimurium, Fur ne semble pas impliqué dans l’interaction avec les macrophages. De plus, les petits ARN RfrA et RfrB sont importants pour la multiplication intracellulaire de Salmonella dans les macrophages pour les deux sérovars. Finalement, la protéine Fur et les petits ARN RfrA et RfrB régulent l’expression de l’opéron fimbriaire tcf, absent du génome de S. Typhimurium. Un site de liaison putatif de la protéine Fur a été identifié dans la région promotrice de tcfA chez S. Typhi, mais une régulation directe n’a pas été confirmée. L’expression de tcf est induite par le fer et par Fur, et est inhibée par les petits ARN RfrA et RfrB. Ainsi, ces régulateurs affectent des gènes de virulence qui sont retrouvés spécifiquement chez S. Typhi. En somme, ce projet a permis de démontrer que les régulateurs de l’homéostasie du fer de Salmonella peuvent affecter la résistance de cette bactérie pathogène à différents stress, notamment le stress oxydatif, la croissance en conditions de carence en fer ainsi que la virulence. Ces régulateurs jouent un rôle distinct chez les sérovars Typhi et Typhimurium. / Regulation of iron homeostasis is crucial for bacteria. For Salmonella, proper timing of the expression of iron acquisition and metabolism genes is important for survival and virulence. This regulation is mediated by the protein Fur and the small non-coding RNAs (sRNAs) RfrA and RfrB. The role of these regulators is to assure that the iron level is high enough for survival and metabolism of Salmonella, and low enough to avoid the toxic effect of iron in the presence of oxygen. Thus far, information on the role of these regulators was principally obtained by studying S. Typhimurium, a generalist serovar causing gastro-enteritis in humans. Very little is known about the role of these regulators in S. Typhi, a human-specific serovar which causes typhoid fever. The goal of this study was to determine the roles of Fur, RfrA and RfrB in iron homeostasis and virulence of Salmonella, and to determine if they have a distinct implication in the serovars Typhimurium and Typhi. First, Fur, RfrA and RfrB regulate iron homeostasis in Salmonella. The results of this study have shown that Fur is required for resistance to oxidative stress and for optimal growth in different in vitro conditions. The sensitivity of the fur mutant is due to the expression of the sRNAs RfrA and RfrB, and this sensitivity is worse in S. Typhi than in S. Typhimurium. Also, Fur represses the transcription of the genes encoding siderophores in high-iron conditions, and the sRNAs RfrA and RfrB are required for enterobactin and salmochelins production in S. Typhi in low-iron conditions. Secondly, these regulators affect the virulence of Salmonella. Fur is important for the motility of Salmonella, especially in S. Typhi. Fur is required for the invasion of both serovars in epithelial cells, and for the uptake and survival of S. Typhi in macrophages. In S. Typhimurium, Fur is not required for the interaction with macrophages. Moreover, the sRNAs RfrA and RfrB are important for the intracellular multiplication of Salmonella within macrophages for both serovars. Finally, the Fur protein and the sRNAs RfrA and RfrB regulate the expression of the tcf fimbrial operon, absent from the genome of S. Typhimurium. A putative Fur binding site was identified in the tcfA promoter region of S. Typhi, but direct regulation has not been confirmed. tcf expression is activated by iron and Fur, and is inhibited by the sRNAs RfrA and RfrB. Therefore, these regulators affect virulence genes that are found specifically in S. Typhi. To conclude, this project demonstrates that the iron homeostasis regulators of Salmonella can affect the bacterial resistance to different stresses, espacially oxidative stress, the growth in iron-limiting conditions and virulence. These regulators have a distinct role in the serovars Typhi and Typhimurium.

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci. / QC 20100723

Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased  replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations.

No description available.

La régulation de l’homéostasie du fer est cruciale chez les bactéries. Chez Salmonella, l’expression des gènes d’acquisition et du métabolisme du fer au moment approprié est importante pour sa survie et sa virulence. Cette régulation est effectuée par la protéine Fur et les petits ARN non codants RfrA et RfrB. Le rôle de ces régulateurs est d’assurer que le niveau de fer soit assez élevé pour la survie et le métabolisme de Salmonella, et assez faible pour éviter l’effet toxique du fer en présence d’oxygène. Les connaissances concernant le rôle de ces régulateurs ont été principalement obtenues par des études chez S. Typhimurium, un sérovar généraliste causant une gastro-entérite chez les humains. Très peu d’informations sont connues sur le rôle de ces régulateurs chez S. Typhi, un sérovar humain-spécifique responsable de la fièvre typhoïde. Le but de cette étude était de déterminer les rôles de Fur, RfrA et RfrB dans l’homéostasie du fer et la virulence de Salmonella, et de démontrer qu’ils ont une implication distincte chez les sérovars Typhi et Typhimurium. Premièrement, Fur, RfrA et RfrB régulent l’homéostasie du fer de Salmonella. Les résultats de cette étude ont démontré que Fur est requis pour la résistance au stress oxydatif et pour une croissance optimale dans différentes conditions in vitro. La sensibilité du mutant fur est due à l’expression des petits ARN RfrA et RfrB, et cette sensibilité est beaucoup plus importante chez S. Typhi que chez S. Typhimurium. Également, Fur inhibe la transcription des gènes codant pour les sidérophores en conditions riches en fer, tandis que les petits ARN RfrA et RfrB semblent être importants pour la production d’entérobactine et de salmochélines chez S. Typhi lors de conditions pauvres en fer. Ensuite, ces régulateurs affectent la virulence de Salmonella. Fur est important pour la motilité de Salmonella, particulièrement chez S. Typhi. Fur est nécessaire pour l’invasion des deux sérovars dans les cellules épithéliales, et pour l’entrée et la survie de S. Typhi dans les macrophages. Chez S. Typhimurium, Fur ne semble pas impliqué dans l’interaction avec les macrophages. De plus, les petits ARN RfrA et RfrB sont importants pour la multiplication intracellulaire de Salmonella dans les macrophages pour les deux sérovars. Finalement, la protéine Fur et les petits ARN RfrA et RfrB régulent l’expression de l’opéron fimbriaire tcf, absent du génome de S. Typhimurium. Un site de liaison putatif de la protéine Fur a été identifié dans la région promotrice de tcfA chez S. Typhi, mais une régulation directe n’a pas été confirmée. L’expression de tcf est induite par le fer et par Fur, et est inhibée par les petits ARN RfrA et RfrB. Ainsi, ces régulateurs affectent des gènes de virulence qui sont retrouvés spécifiquement chez S. Typhi. En somme, ce projet a permis de démontrer que les régulateurs de l’homéostasie du fer de Salmonella peuvent affecter la résistance de cette bactérie pathogène à différents stress, notamment le stress oxydatif, la croissance en conditions de carence en fer ainsi que la virulence. Ces régulateurs jouent un rôle distinct chez les sérovars Typhi et Typhimurium. / Regulation of iron homeostasis is crucial for bacteria. For Salmonella, proper timing of the expression of iron acquisition and metabolism genes is important for survival and virulence. This regulation is mediated by the protein Fur and the small non-coding RNAs (sRNAs) RfrA and RfrB. The role of these regulators is to assure that the iron level is high enough for survival and metabolism of Salmonella, and low enough to avoid the toxic effect of iron in the presence of oxygen. Thus far, information on the role of these regulators was principally obtained by studying S. Typhimurium, a generalist serovar causing gastro-enteritis in humans. Very little is known about the role of these regulators in S. Typhi, a human-specific serovar which causes typhoid fever. The goal of this study was to determine the roles of Fur, RfrA and RfrB in iron homeostasis and virulence of Salmonella, and to determine if they have a distinct implication in the serovars Typhimurium and Typhi. First, Fur, RfrA and RfrB regulate iron homeostasis in Salmonella. The results of this study have shown that Fur is required for resistance to oxidative stress and for optimal growth in different in vitro conditions. The sensitivity of the fur mutant is due to the expression of the sRNAs RfrA and RfrB, and this sensitivity is worse in S. Typhi than in S. Typhimurium. Also, Fur represses the transcription of the genes encoding siderophores in high-iron conditions, and the sRNAs RfrA and RfrB are required for enterobactin and salmochelins production in S. Typhi in low-iron conditions. Secondly, these regulators affect the virulence of Salmonella. Fur is important for the motility of Salmonella, especially in S. Typhi. Fur is required for the invasion of both serovars in epithelial cells, and for the uptake and survival of S. Typhi in macrophages. In S. Typhimurium, Fur is not required for the interaction with macrophages. Moreover, the sRNAs RfrA and RfrB are important for the intracellular multiplication of Salmonella within macrophages for both serovars. Finally, the Fur protein and the sRNAs RfrA and RfrB regulate the expression of the tcf fimbrial operon, absent from the genome of S. Typhimurium. A putative Fur binding site was identified in the tcfA promoter region of S. Typhi, but direct regulation has not been confirmed. tcf expression is activated by iron and Fur, and is inhibited by the sRNAs RfrA and RfrB. Therefore, these regulators affect virulence genes that are found specifically in S. Typhi. To conclude, this project demonstrates that the iron homeostasis regulators of Salmonella can affect the bacterial resistance to different stresses, espacially oxidative stress, the growth in iron-limiting conditions and virulence. These regulators have a distinct role in the serovars Typhi and Typhimurium.

OCL1 (Outer Cell Layer 1) est le membre fondateur, chez le maïs, de la famille multigénique regroupant les facteurs de transcription HD-ZIP IV. La plupart de ces gènes s’exprime préférentiellement dans l’épiderme, et chez Arabidopsis l’étude de mutants a montré que certains HD-ZIP IV étaient essentiels pour la différenciation de cette couche cellulaire. Lors de ma thèse je me suis intéressée à la régulation du gène OCL1 par un petit ARN non codant. En effet, la conservation au sein des 3’UTR de plusieurs gènes HD-ZIP IV d’un motif de 21 nucléotides (nt) suggérait l’existence d’un tel mécanisme. J’ai mis en évidence que ce motif de 21 nt était conservé des Bryophytes aux Angiospermes et qu’il était toujours couplé à un second motif conservé d’une taille de 19 nt avec lequel il peut s’apparier pour former une structure secondaire de type tige-boucle. J’ai démontré l’existence d’un petit ARN ayant une séquence (quasi) complémentaire au site de 21 nt. La biogenèse de ce petit ARN de 24 nt que nous avons nommé small1, dépend de RDR2/ MOP1, DCL3 et Pol IV/ RMR6, composants normalement requis pour le mécanisme de RdDM. A l’aide d’un système GFP sensor, j’ai cependant mis en évidence que small1 régulait l’expression de son gène cible par inhibition de la traduction et non par RdDM. Ces expériences ont par ailleurs démontré qu’OCL1 n’est pas régulé uniquement par small1, mais également via un second mécanisme dans lequel pourrait intervenir la structure secondaire de type tige-boucle. Enfin, j’ai montré que small1 possède une extrémité 5’modifiée, expliquant ainsi son absence des banques de données et définissant aussi une nouvelle classe de petits ARN chez les plantes. / Small non-coding RNAs are versatile riboregulators that control gene expression at the transcriptional or post-transcriptional level, governing many facets of plant development and stress responses. We previously suggested the possible regulation of OCL1 (Outer Cell Layer1) by a small RNA based on the intriguing presence of two conserved motives of 19 and 21nt in its 3’UTR. ZmOCL1 is a founding member of the HD-ZIP IV gene family encoding plant specific transcription factors mainly involved in epidermis differentiation and specialization. Here we present evidence for the existence of a 24 nt small RNA complementary to ZmOCL1 3’UTR which accumulates preferentially in maize reproductive organs but also in Arabidopsis flowers and inflorescences. The biogenesis of this 24 nt small RNA (that we named small1) depends on MOP1/RDR2 and RMR6/POLIV and DCL3, components normally required for RNA-dependent DNA-methylation. Unexpectedly GFP-sensor experiments showed that small1 may regulate its target at the post-transcriptional level, mainly through translational inhibition. These experiments further highlighted the importance of additional 3’UTR sequences required for efficient target repression, possibly implicating a secondary stem-loop structure. Finally, we showed that small1 is modified at its 5’ end, which not only explains its absence from the current databases but also defines a novel class of plant small RNAs.

Argonaute proteins are small DNA/RNA-guided endonucleases found in all domains of life. In animals, small RNAs of length 21–35 nucleotides direct the PIWI-clade of Argonautes to silence complementary target RNAs; these are called PIWI-interacting RNAs (piRNAs). During spermatogenesis in mice, piRNA-guided PIWI proteins, MIWI2, MILI, and MIWI, silence transposons, regulate expression of protein-coding genes and are necessary for fertility. A working endonuclease activity of MIWI and MILI is essential to complete spermatogenesis. Yet, both MIWI and MILI produce weak and slow target cleavage in vitro, thwarting biochemical examination of the silencing step. Here, we find that PIWI proteins require an auxiliary protein to efficiently cleave their targets, unlike any other known Argonaute. Gametocyte Specific Factor 1 (GTSF1) is a conserved zinc-finger protein essential for fertility and piRNA-directed silencing. We show GTSF1 accelerates the pre-steady-state rate of target cleavage by MIWI and MILI; this role of GTSF1 is also preserved in insects. A critical step in GTSF1 mechanism entails binding RNA. GTSF1 allowed detailed kinetic analyses of catalytic PIWIs: they require extensive 3′ complementarity between the guide and target to efficiently cleave them, but this base-pairing also limits turnover. Interestingly, within a species, different PIWI proteins have unique kinetic properties. In sum, our findings provide molecular mechanisms of GTSF1 function and target silencing by PIWIs as well as a useful method for future studies.