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Phytoene synthase (PSY) converts two molecules of geranyl-geranyl diphosphate to phytoene, the key regulatory step in carotenogenesis. However, post-translational mechanisms that control PSY expression are scarcely understood. Carotenoid biosynthesis (mainly bacterioruberin) is a distinctive feature of haloarchaea thriving in hypersaline environments. Carotenogenesis is negatively regulated by the AAA+ LonB protease in the haloarchaeon Haloferax volcanii as it controls PSY degradation. We investigated the relevance of the C-terminal portion of HvPSY as a regulatory element for carotenoid biosynthesis. H. volcanii mutants were constructed to express full-length HvPSY protein (strain HVPSYwt) and truncated HvPSY lacking 10 (HVPSY10), 20 (HVPSY20) or 34 amino acids (HVPSY34) at the C-terminus. Cells of HVPSY20 and HVPSY34 showed hyperpigmentation (bacterioruberin content 3-fold higher than HVPSYwt) which correlated with increased PSY protein abundance (2-fold in HVPSY34) while they contained less psy transcript level compared with HVPSYwt. In vivo degradation assays showed that HvPSY34 was more stable than HvPSYwt. Collectively, these results show that the C-terminal region of HvPSY contains a ‘recognition determinant’ for proteolysis in H. volcanii. Preliminary evidence suggests that LonB is involved in the recognition mechanism. This study provides the first identification of a regulatory sequence in an archaeal PSY for the post-translational control of carotenogenesis.

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further showed that the use of inteins expands functional sequence space for splitting a protein. We also demonstrated the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins. Furthermore, the intein inserted at an identified site could be engineered by the transposon again to become partially chemically inducible, and to some extent enabled post-translational tuning on host protein function. Our work offers a generalizable and systematic route towards creating split protein-intein fusions and conditional inteins for protein activity control.

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology., Split inteins are powerful tools for designing synthetic split proteins. Here the authors use a mini-Mu transposon screen to map split sites, enabling the development of protein-based logic gates and fine control of protein activity.

Mu transposition-based cloning of DNA circles employs in vitro transposition reaction to deliver both the plasmid origin of replication and a selectable marker into the target DNA of interest. We report here the construction of a platform for the purpose that contains ten mini-Mu transposons with five different replication origins, enabling a variety of research approaches for the discovery and study of circular DNA. We also demonstrate that the simultaneous use of two transposons, one with the origin of replication and the other with selectable marker, is beneficial as it improves the cloning efficiency by reducing the fraction of autointegration-derived plasmid clones. The constructed transposons now provide a set of new tools for the studies on DNA circles and widen the applicability of Mu transposition based approaches to clone circular DNA from various sources.

Halophilic archaea thrive in hypersaline conditions associated with desiccation, ultraviolet (UV) irradiation and redox active compounds, and thus are naturally tolerant to a variety of stresses. Here, we identified mutations that promote enhanced tolerance of halophilic archaea to redox-active compounds using Haloferax volcanii as a model organism. The strains were isolated from a library of random transposon mutants for growth on high doses of sodium hypochlorite (NaOCl), an agent that forms hypochlorous acid (HOCl) and other redox acid compounds common to aqueous environments of high concentrations of chloride. The transposon insertion site in each of twenty isolated clones was mapped using the following: (i) inverse nested two-step PCR (INT-PCR) and (ii) semi-random two-step PCR (ST-PCR). Genes that were found to be disrupted in hypertolerant strains were associated with lysine deacetylation, proteasomes, transporters, polyamine biosynthesis, electron transfer, and other cellular processes. Further analysis revealed a Delta psmA1 (alpha 1) markerless deletion strain that produces only the alpha 2 and beta proteins of 20S proteasomes was hypertolerant to hypochlorite stress compared with wild type, which produces alpha 1, alpha 2, and beta proteins. The results of this study provide new insights into archaeal tolerance of redox active compounds such as hypochlorite.

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

The capacity of transposable elements to insert into the genomes has been harnessed during the past decades to various in vitro and in vivo applications. This chapter describes in detail the general protocols and principles applicable for the Mu in vitro transposition reaction as well as the assembly of DNA transposition complexes that can be electroporated into bacterial cells to accomplish efficient gene delivery. These techniques with their modifications potentiate various gene and genome modification applications, which are discussed briefly here, and the reader is referred to the original publications for further details.

The capacity of transposable elements to insert into the genomes has been harnessed during the past decades to various in vitro and in vivo applications. This chapter describes in detail the general protocols and principles applicable for the Mu in vitro transposition reaction as well as the assembly of DNA transposition complexes that can be electroporated into bacterial cells to accomplish efficient gene delivery. These techniques with their modifications potentiate various gene and genome modification applications, which are discussed briefly here, and the reader is referred to the original publications for further details.

Archaea constitute the third domain of life, but studies on their physiology and other features have lagged behind bacteria and eukarya, largely due to the challenging biology of archaea and concomitant difficulties in methods development. The use of genome-wide en masse insertion mutagenesis is one of the most efficient means to discover the genes behind various biological functions, and such a methodology is described in this chapter for a model archaeon Haloferax volcanii. The strategy successfully employs efficient in vitro transposition in combination with gene targeting in vivo via homologous recombination. The methodology is general and should be transferable to other archaeal species.

Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA transposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantifies transpositional recombination efficiency and is based on an in vitro transposition reaction with a target plasmid carrying a lethal ccdB gene. If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other transposition-based systems with a functional in vitro reaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications.

Polintons are a recently discovered group of large transposable elements (

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella—structures required for motility—share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air–liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876—also critical for both motility and adhesion—will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects.

Archaea constitute the third domain of life, but studies on their physiology and other features have lagged behind bacteria and eukarya, largely due to the challenging biology of archaea and concomitant difficulties in methods development. The use of genome-wide en masse insertion mutagenesis is one of the most efficient means to discover the genes behind various biological functions, and such a methodology is described in this chapter for a model archaeon Haloferax volcanii. The strategy successfully employs efficient in vitro transposition in combination with gene targeting in vivo via homologous recombination. The methodology is general and should be transferable to other archaeal species.

We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion., Sec1p/Munc18 (SM) family proteins regulate SNARE complex function in membrane fusion through their interactions with syntaxins. In addition to syntaxins, only a few SM protein interacting proteins are known and typically, their binding modes with SM proteins are poorly characterized. We previously identified Mso1p as a Sec1p-binding protein and showed that it is involved in membrane fusion regulation. Here we demonstrate that Mso1p and Sec1p interact at sites of exocytosis and that the Mso1p–Sec1p interaction site depends on a functional Rab GTPase Sec4p and its GEF Sec2p. Random and targeted mutagenesis of Sec1p, followed by analysis of protein interactions, indicates that Mso1p interacts with Sec1p domain 1 and that this interaction is important for membrane fusion. In many SM family proteins, domain 1 binds to a N-terminal peptide of a syntaxin family protein. The Sec1p-interacting syntaxins Sso1p and Sso2p lack the N-terminal peptide. We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding, and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion.

Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn 916 -based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens , which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens .

Transposons provide useful tools for genetics and genomics studies, as they can be modified easily for a variety of purposes. In this study, a strategy to clone circular DNA was developed on the basis of an efficient Mu in vitro transposition reaction catalyzed by MuA transposase. The transposon used contains a selectable marker as well as an origin of replication, and in vitro integration of the transposon into circular DNA generates a plasmid that can replicate in E. coli. We show that the substrate stoichiometry plays an important role in the profile of intermolecular versus intramolecular transposition reaction products. Increasing the relative amount of target DNA reduced the frequency of intramolecular products that are non-productive with regard to the developed cloning application. Such autointegration was also reduced in the reactions containing phage Mu-encoded MuB, indicating that this protein can be used for cloning in combination with MuA, and it is particularly useful with a limited amount of target DNA. The developed strategy can now be utilized to clone DNA circles regardless of their origin as long as their size is not prohibitive for transformation.

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

Bacteriophage Mu uses non-replicative transposition for integration into the host's chromosome and replicative transposition for phage propagation. Biochemical and structural comparisons together with evolutionary considerations suggest that the Mu transposition machinery might share functional similarities with machineries of the systems that are known to employ a hairpin intermediate during the catalytic steps of transposition. Model transposon end DNA hairpin substrates were used in a minimal-component in vitro system to study their proficiency to promote Mu transpososome assembly and subsequent MuA-catalyzed chemical reactions leading to the strand transfer product. MuA indeed was able to assemble hairpin substrates into a catalytically competent transpososome, open the hairpin ends and accurately join the opened ends to the target DNA. The hairpin opening and transposon end cleavage reactions had identical metal ion preferences, indicating similar conformations within the catalytic center for these reactions. Hairpin length influenced transpososome assembly as well as catalysis: longer loops were more efficient in these respects. In general, MuA's proficiency to utilize different types of hairpin substrates indicates a certain degree of flexibility within the transposition machinery core. Overall, the results suggest that non-replicative and replicative transposition systems may structurally and evolutionarily be more closely linked than anticipated previously.

Background Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways. Results Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. Conclusions We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea. Electronic supplementary material The online version of this article (doi:10.1186/s12915-014-0103-3) contains supplementary material, which is available to authorized users.

Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between myotilin and actin. Functionally important regions in myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.

Hyperpolarizing fast inhibitory neurotransmission by gamma-aminobutyric acid and glycine requires an efficient chloride extrusion mechanism in postsynaptic neurons. A major effector of this task in adult animals is the potassium-chloride co-transporter KCC2 that is selectively and abundantly expressed postsynaptically in most CNS neurons. Yet, the role of KCC2 in adult brain at the systems level is poorly known. Here, we characterize the behaviour of mice doubly heterozygous for KCC2 null and hypomorphic alleles that retain 15-20% of normal KCC2 protein levels in the brain. These hypomorphic KCC2-deficient mice were viable and fertile but weighed 15-20% less than wild-type littermates at 2 weeks old and thereafter. The mice displayed increased anxiety-like behaviour in several tests including elevated plus-maze and were more susceptible to pentylenetetrazole-induced seizures. Moreover, the mice were impaired in water maze learning and showed reduced sensitivity to tactile and noxious thermal stimuli in von Frey hairs, hot plate and tail flick tests. In contrast, the mice showed normal spontaneous locomotor activity in open field and Y-maze tests, and intact motor coordination in rotarod and beam tests. The results suggest that requirements for KCC2-dependent fast hyperpolarizing inhibition may differ among various functional systems of the CNS. As shunting inhibition is expected to be intact in KCC2-deficient neurons, these mice may provide a useful tool to study the specific functions and relative importance of hyperpolarizing fast synaptic inhibition in adult CNS that may have implications for human neuropsychiatric disorders, such as epilepsy, pain and anxiety.

Protein structure-function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins. Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the alpha-complementing domain 1 of Escherichia coli beta-galactosidase, yielding mutants at 100% efficiency. Each mutant contained an accurate 15-bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence. Individual mutants (alpha-donors) were analyzed for their ability to restore (by alpha-complementation) beta-galactosidase activity of the M15 deletion mutant (alpha-acceptor), and the data were correlated to the structure of the beta-galactosidase tetramer. Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior. Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance. Inhibitory insertions within the N-terminus and loop regions most likely influenced protein tetramerization via direct local effects on protein-protein interactions. Within the domain 1 core, the insertions probably caused either lateral shifting of the polypeptide chain toward the protein's exterior or produced more pronounced structural distortions. Six percent of the mutant proteins exhibited temperature sensitivity, in general suggesting the method's usefulness for generation of conditional phenotypes. The method should be applicable to any cloned protein-encoding gene.

The Mu transpositional DNA recombination machinery selects target sites by assembling a protein-DNA complex that interacts with the target DNA and reacts whenever it locates a favorable sequence composition. Splicing of a transposon into the target generates a 5-bp duplication that reflects the original target site. Preferential usage of different target pentamers was examined with a minimal Mu in vitro system and quantitatively compiled consensus sequences for the most preferred and the least preferred sites were generated. When analyzed as base steps, preferences toward certain steps along the 5-bp target site were detected. We further show that insertion sites can be predicted on the basis of additively calculated base step values. Also surrounding sequences influence the preference of a given pentamer; a symmetrical structural component was revealed, suggesting potential hinges at and around the target site.

We present a detailed and quantitative analysis of the functional characteristics of the 1,000-nucleotide segment at the 5′ end of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This segment of the viral genome contains several important cis -acting sequences, including the TAR, polyadenylation, viral att site, minus-strand primer-binding site, and 5′ splice donor sequences, as well as coding sequences for the matrix protein and the N-terminal half of the capsid protein. The genetic footprinting technique was used to determine quantitatively the abilities of 134 independent insertion mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete reverse transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were constructed and analyzed en masse, greatly decreasing the labor typically involved in mutagenesis studies. The results confirmed the presence of several previously known functional features in this region of the HIV genome and provided evidence for several novel features, including newly identified cis -acting sequences that appeared to contribute to (i) the formation of stable viral transcripts, (ii) viral RNA packaging, and (iii) an early step in viral replication. The results also pointed to an unanticipated trans -acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcripts. Finally, in contrast to previous reports, the results of this study suggested that detrimental mutations in the matrix and capsid proteins principally interfered with viral assembly.

During the last decade, the sequenced DNA information has accumulated with increasing pace. The yeast genome, as well as a variety of microbial genomes, has already been sequenced entirely, and projects destined to sequence several eukaryotic genomes including those of man, mouse, fly, worm, and plant are being completed in the near future (Ash 1997; Koonin 1997; Beck and Sterk 1998). Nevertheless, the need for DNA sequencing is not expected to be diminished. For example, comparative genome analyses, between species or within species, that will yield a wealth of information concerning principles of life, will necessarily involve massive DNA sequencing. Efficient DNA sequencing strategy that will yield sequence information reliably and rapidly is a necessity for a sequencing project. In principle, the strategies used so far can be divided into three main categories: (1) random, (2) directed, and (3) transposon-based strategies. The most widely used strategy, especially for large-scale DNA sequencing projects, is a random shotgun approach (for review, see Hunkapiller et al. 1992; Fraser and Fleischmann 1997). Directed strategies, typified by primer-walking and nested deletions, have been used mainly to sequence DNA regions of moderate size (for review, see Ansorge et al. 1997). Transposon-based strategies combine features of random and directed approaches and can be used efficiently in various types of sequencing projects. Several DNA sequencing strategies based on in vivo DNA transposition reactions have been characterized (Adachi et al. 1987; Phadnis et al. 1989; Strathman et al. 1991; Kasai et al. 1992; Berg et al. 1993). However, in vivo approaches require special host strains and multiple steps of manipulation that limit the usefulness of these strategies. In addition, in some of the cases, a stringency of the target-site selectivity was not optimal for sequencing purposes. In vitro DNA transposition-based sequencing strategy has also been described and shown to be highly useful, especially for sequencing repetitive DNA (Devine and Boeke 1994; Devine et al. 1997). The system utilized a customized artificial transposon in combination with integrase activity present in purified yeast Ty1 retrotransposon virus-like particles. However, no DNA sequencing strategy that would utilize a single transposase protein in in vitro conditions has yet been described. Such a system would be advantageous: Relative simplicity of protein purification compared with that of protein complexes, as well as at least potentially more preferable stability characteristics of proteins, make a transposase-catalyzed reaction an attractive choice for sequencing purposes. Mu DNA transposition reaction is one of the best-characterized transposition reactions (Mizuuchi 1992 a,b). In vivo, and in certain reaction conditions with plasmid substrates in vitro, the reaction is relatively complex and involves several protein and DNA cofactors (for reviews, see Chaconas et al. 1996; Lavoie and Chaconas 1996) of which the phage-encoded MuB protein affects target selection (Adzuma and Mizuuchi 1988, 1989; Mizuuchi and Mizuuchi 1993). However, with certain DNA substrates and reaction conditions in vitro the requirements for efficient transposition reaction are substantially relaxed (Craigie and Mizuuchi 1986, 1987; Mizuuchi and Mizuuchi 1989; Baker and Mizuuchi 1992; Savilahti et al. 1995). The minimal macromolecular components for in vitro transposition into intermolecular targets are the MuA transposase, the donor DNA, and the target DNA (Savilahti et al. 1995). The chemical steps of Mu transposition proceed within a protein–DNA complex called Mu transpososome that in its core contains the critical components, a tetramer of MuA transposase and Mu DNA ends (Craigie and Mizuuchi 1987; Surette et al. 1987; Lavoie et al. 1991; Baker and Mizuuchi 1992; Baker et al. 1993; Savilahti et al. 1995). Initially, the Mu transpososome core is assembled from four molecules of MuA and two Mu DNA ends, and this underlining architecture is then maintained throughout the two chemical steps of transposition. First, the transposon DNA is cleaved at its 3′ ends (called the donor cleavage). Second, the target DNA is cut at staggered positions and the target 5′ ends are joined into the 3′ ends of the transposon DNA in a concerted reaction (called strand transfer). In vitro, the first of the chemical steps can also be bypassed artificially by use of a modified donor DNA substrate. It was shown that Mu donor DNA that was cut precisely at Mu 3′ ends by a restriction endonuclease was efficiently utilized in transferring these precut 3′ ends into the target DNA (Craigie and Mizuuchi 1987). In turn, this result brought about an opportunity to use the Mu transposition reaction in a simple manner for genetic manipulation. In this paper we describe a highly efficient and easy methodology for DNA sequencing that is based on the bacteriophage Mu in vitro transposition reaction. As a test case, we used the system to determine a 10,288-bp sequence from a mouse genomic locus containing a Kcc2 gene. The respective protein, KCC2, is a K+/Cl− cotransporter that is abundantly expressed in brain and suggested to be involved in extruding chloride in mature neurons (Payne et al. 1996). We are currently producing mice with targeted inactivation of Kcc2 and, for this purpose, the sequence of the mouse genomic fragment was needed. Bacteriophage Mu in vitro DNA transposition system proved to be very useful for DNA sequencing purposes: (1) The reaction in vitro is simple; only one protein component, the MuA transposase, is needed in addition to donor DNA, target DNA, and buffer. (2) The transposition reaction is highly efficient and reproducible in in vitro conditions. (3) The artificial cat–Mu transposon contains an easily selectable marker, chloramphenicol acetyltransferase gene (cat), that allows convenient selection of integrants. (4) Mu in vitro transposition produces relatively even distribution of integrations into the target DNA.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general. Public Library of Science open access

Background: Mu transposase (MuA) is a multidomain protein encoded by the bacteriophage Mu genome. It is responsible for translocation of the Mu genome, which is the largest and most efficient transposon known. While the various domains of MuA have been delineated by means of biochemical methods, no data have been obtained to date relating to its tertiary structure. Results We have solved the three–dimensional solution structure of the DNA–binding domain (residues 1–76; MuA 76 ) of MuA by multidimensional heteronuclear NMR spectroscopy. The structure consists of a three–membered α –helical bundle buttressed by a three–stranded antiparallel β –sheet. Helices H1 and H2 and the seven–residue turn connecting them comprise a helix–turn–helix (HTH) motif. In addition, there is a long nine–residue flexible loop or wing connecting strands B2 and B3 of the sheet. NMR studies of MuA 76 complexed with a consensus DNA site from the internal activation region of the Mu genome indicate that the wing and the second helix of the HTH motif are significantly perturbed upon DNA binding. Conclusion While the general appearance of the DNA–binding domain of MuA 76 is similar to that of other winged HTH proteins, the connectivity of the secondary structure elements is permuted. Hence, the fold of MuA 76 represents a novel class of winged HTH DNA–binding domain.

MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.

Background Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host. Results When mature Peb1 was expressed without its SecA-YEG -dependent signal sequence and without the putative signal peptidase II recognition sequence in E. coli MKS111ΔHBB lacking the flagellar secretion complex, the protein was found in the periplasm and growth medium which indicated a flagellum-independent translocation. We assessed the Peb1 secretion proficiency by an exhaustive search for transport-affecting regions using a transposition-based scanning mutagenesis strategy. Strikingly, insertion mutagenesis of only two segments, called TAR1 (residues 42 and 43) and TAR2 (residues 173 to 180), prevented Peb1 secretion individually. We confirmed the importance of TAR regions by subsequent site-specific mutagenesis and verified that the secretion deficiency of Peb1 mutants was not due to insolubility or aggregation of the proteins in the cytoplasm. We found by cell fractionation that the mutant proteins were present in the periplasm as well as in the cytoplasm of MKS12. Hence, mutagenesis of TAR regions did not affect export of Peb1 across the cytoplasmic membrane, whereas its export over the outer membrane was markedly impaired. Conclusions We propose that the localization of the model protein Peb1 in the growth medium of E. coli is due to active secretion by a still unknown pathway of E. coli. The secretion apparently is a two-step process involving a periplasmic step and the TAR regions.

Background Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition. Results Here we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. Conclusions The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.

Using electroporation with the phage PRD1 genome, we set up a high-frequency DNA transfer system for a linear dsDNA molecule with 5'-covalently linked terminal proteins. The transfer was saturated when more than 100 ng of PRD1 genome was used. Electroporation efficiency was about four orders of magnitude higher than that obtained with transfection. Removal of the terminal protein abolished plaque formation, which could not be rescued by supplying the terminal protein or phage DNA polymerase or both in trans.

High-precision mapping of regions involved in protein-protein interfaces of interacting protein partners is an essential component on a path to understand various cellular functions. Transposon-based systems, particularly those involving in vitro reactions, offer exhaustive insertion mutant libraries and high-throughput platforms for many types of genetic analyses. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision that is based on transposition-assisted construction, sampling, and analysis of a comprehensive insertion mutant library. The methodology integrates random pentapeptide mutagenesis of proteins, yeast two-hybrid screening, and high-resolution genetic footprinting. This straightforward strategy is general, and it provides a rapid and easy means to identify critical contact regions in proteins without the requirement of prior structural knowledge.

Conditional gene targeting strategies are progressively used to study gene function tissue-specifically and/or at a defined time period. Instrumental to all of these strategies is the generation of targeting vectors, and any methodology that would streamline the procedure would be highly beneficial. We describe a comprehensive transposition-based strategy to produce gene-targeting vectors for the generation of mouse conditional alleles. The system employs a universal cloning vector and two custom-designed mini-Mu transposons. It produces targeting constructions directly from BAC clones, and the alleles generated are modifiable by Cre and Flp recombinases. We demonstrate the applicability of the methodology by modifying two mouse genes, Chd22 and Drapc1. This straightforward strategy should be readily suitable for high-throughput targeting vector production.

Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY , srrA , mgrA , and fmtA , a putative DEAD-box helicase, two members of the zinc-metallo-β lactamase/β-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis . Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non- ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non- ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

We developed a novel strategy based on in vitro DNA transposition of phage Mu to construct vectors for "knock-in" of the gene encoding Cre recombinase into endogenous loci in embryonic stem cells. This strategy was used to introduce Cre into the mouse Meox1 locus, which was expected to drive Cre expression in the presomitic and somitic mesoderm. In embryos heterozygous for both Meox1(Cre) and R26R or Z/AP reporter alleles, specific and efficient recombination of the reporter alleles was detected in the maturing somites and their derivatives, including developing vertebrae, skeletal muscle, back dermis, as well as endothelium of the blood vessels invading the spinal cord and developing limbs. In contrast to the somitic mesoderm, Cre activity was not observed in the cranial paraxial mesoderm. Thus, the Meox1(Cre) allele allows detailed fate-mapping of Meox1-expressing tissues, including derivatives of the somitic mesoderm. We used it to demonstrate dynamic changes in the composition of the mesenchyme surrounding the developing inner ear. Meox1(Cre) may also be used for tissue-specific mutagenesis in the somitic mesoderm and its derivatives.

Transposon mutagenesis is a powerful technique for generating collections of insertion mutants for genetic studies. This paper describes how phage Mu DNA transposition complexes, transpososomes, can be exploited for gene delivery to efficiently introduce selectable markers to genomes of Gram-positive bacteria. Mu transpososomes were assembled in vitro with custom-designed mini-Mu transposons, concentrated, and electroporated into cells of three Gram-positive bacterial species: Staphylococcus aureus, Streptococcus pyogenes and Streptococcus suis. Within cells, the complexes reproduced an authentic DNA transposition reaction and integrated the delivered transposons into the bacterial genomes, yielding single-copy insertions. The integration efficiency among different species and strains of Gram-positive bacteria ranged from 1x10(1) to 2x10(4) c.f.u. (mug introduced transposon DNA)(-1). The strategy should be applicable to a variety of other Gram-positive species after initial optimization of certain key factors affecting transposon delivery, such as the preparation method of competent cells and physical parameters of electroporation. This study extends the scope of the Mu transpososome delivery-based genomic DNA integration strategy to Gram-positive bacteria. Thus, a straightforward generation of sizeable mutant banks is feasible for these bacteria, potentiating several types of genomic-level approaches for studies of a variety of important bacterial processes, such as pathogenicity.

Bacteriophage YeO3-12 is a T7/T3-related lytic phage that naturally infects Yersinia enterocolitica serotype O:3 strains by using the lipopolysaccharide O polysaccharide (O antigen) as its receptor. The phage genome is a 39,600-bp-long linear, double-stranded DNA molecule that contains 58 genes. The roles of many of the genes are currently unknown. To identify nonessential genes, the isolated phage DNA was subjected to MuA transposase-catalyzed in vitro transposon insertion mutagenesis with a lacZ gene-containing reporter transposon. Following electroporation into Escherichia coli DH10B and subsequent infection of E. coli JM109/ pAY100, a strain that expresses the Y. enterocolitica O:3 O antigen on its surface, mutant phage clones were identified by their -galactosidase activity, manifested as a blue color on indicator plates. Transposon insertions were mapped in a total of 11 genes located in the early and middle regions of the phage genome. All of the mutants had efficiencies of plating (EOPs) and fitnesses identical to those of the wild-type phage when grown on E. coli JM109/pAY100. However, certain mutants exhibited altered phenotypes when grown on Y. enterocolitica O:3. Transposon insertions in genes 0.3 to 0.7 decreased the EOP on Y. enterocolitica O:3, while the corresponding deletions did not, suggesting that the low EOP was not caused by inactivation of the genes per se. Instead, it was shown that in these mutants the low EOP was due to the delayed expression of gene 1, coding for RNA polymerase. On the other hand, inactivation of gene 1.3 or 3.5 by either transposon insertion or deletion decreased phage fitness when grown on Y. enterocolitica. These results indicate that YeO3-12 has adapted to utilize Y. enterocolitica as its host and that these adaptations include the products of genes 1.3 and 3.5, DNA ligase and lysozyme, respectively. Yersinia enterocolitica is a gram-negative bacterial species that belongs to the family Enterobacteriaceae. This species includes over 30 known serotypes, a number of which are human pathogens. While the most common pathogenic serotypes in Europe, Canada, Japan, and South Africa are O:3 and O:9, in the United States serotype O:8 is more prevalent. Y. enterocolitica is widely distributed in nature, with swine being the major reservoir of the pathogenic strains (6). YeO3-12 is a lytic bacteriophage that infects Y. enterocolitica serotype O:3 strains (28, 30). The receptor of the phage is Y. enterocolitica O:3 O antigen, and the phage is able to infect and proliferate in Escherichia coli strains that express the Y. enterocolitica O:3 O-antigen gene cluster (2). The genome of YeO3-12 is a 39,600-bp-long linear, double-stranded DNA molecule, whose nucleotide sequence has been completely determined (GenBank accession no. AJ251805). The genome harbors 58 putative genes that all are transcribed from the same DNA strand (30). YeO3-12 belongs to the T7 group of lytic phages, with E. coli phage T3 being its closest relative. The genome organizations of T7, T3, and YeO3-12 are colinear, with early, middle, and late regions, and the overall identity between the YeO3-12 and T3 genomes is 84%. Based on similarity to T7 and T3 genes, probable functions of many YeO3

Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5′-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79–N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector.

As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.

Protein structure-function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins. Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the alpha-complementing domain 1 of Escherichia coli beta-galactosidase, yielding mutants at 100% efficiency. Each mutant contained an accurate 15-bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence. Individual mutants (alpha-donors) were analyzed for their ability to restore (by alpha-complementation) beta-galactosidase activity of the M15 deletion mutant (alpha-acceptor), and the data were correlated to the structure of the beta-galactosidase tetramer. Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior. Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance. Inhibitory insertions within the N-terminus and loop regions most likely influenced protein tetramerization via direct local effects on protein-protein interactions. Within the domain 1 core, the insertions probably caused either lateral shifting of the polypeptide chain toward the protein's exterior or produced more pronounced structural distortions. Six percent of the mutant proteins exhibited temperature sensitivity, in general suggesting the method's usefulness for generation of conditional phenotypes. The method should be applicable to any cloned protein-encoding gene.

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.

Transposon insertions were obtained in early and middle regions of the phage genome only, which is consistent with the essential nature of the late genes. Phage genes coding for DNA ligase and lysozyme were found to be needed for efficient propagation of φYeO3-12 in YeO3-c but not in E. coli. Also, in Yersinia but not in other bacteria tested, phage gene 0.45 seemed to be important in the early stages of infection. Still, further studies are needed to understand the biological basis for these phenotypes.

Screening of phage display libraries allows rapid identification of peptides binding to a target. However, functional analysis of the phage sequences and their reproduction as soluble and stable peptides are often the most time-consuming part in the screening. We have used here intein-based peptide biosynthesis to produce a phage-display derived gelatinase inhibitory peptide CTTHWGFTLC and to identify the critical residues for gelatinase inhibitory activity by performing alanine-scanning mutagenesis. By biosynthetic incorporation of 5-fluorotryptophan, we obtained an inhibitor of MMP-2 and MMP-9 gelatinases that showed a 6-fold enhancement in serum stability in comparison to the wild-type peptide. The new peptide also had an improved ability to inhibit tumor cell migration. These studies indicate the utility of intein methodology for synthesis and design of peptides obtained by phage display.