Site-specific Insertion Research Articles

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

Multivalent site-specific phage modification enhances the binding affinity of receptor ligands.

High-throughput screening of combinatorial chemical libraries is a powerful approach for identifying targeted molecules. The display of combinatorial peptide libraries on the surface of bacteriophages offers a rapid, economical way to screen billions of peptides for specific binding properties and has impacted fields ranging from cancer to vaccine development. As a modification to this approach, we have previously created a system that enables site-specific insertion of selenocysteine (Sec) residues into peptides displayed pentavalently on M13 phage as pIII coat protein fusions. In this study, we show the utility of selectively derivatizing these Sec residues through the primary amine of small molecules that target a G protein-coupled receptor, the adenosine A1 receptor, leaving the other coat proteins, including the major coat protein pVIII, unmodified. We further demonstrate that modified Sec-phage with multivalent bound agonist binds to cells and elicits downstream signaling with orders of magnitude greater potency than that of unconjugated agonist. Our results provide proof of concept of a system that can create hybrid small molecule-containing peptide libraries and open up new possibilities for phage-drug therapies.

Abstract 19831: In Vivo Molecular Imaging of Human Pluripotent Stem Cell-derived Cardiomyocytes in a Murine Myocardial Injury Model via a Safe Harbor Integration of a Reporter Gene

Background: Clinical application of pluripotent stem cells and their tissue specific derivatives will require in vivo monitoring of engraftment and systemic distribution following transplantation. Clinical translation of molecular imaging, employing reporter gene (RG) technology, requires site-specific transgene insertion at safe harbor loci, such as AAVS1. Hypothesis: Zinc finger nuclease (ZFN)-mediated integration of a RG into the AAVS1 locus in human embryonic and induced pluripotent stem cells (hESCs and iPSCs) will allow monitoring of pluripotent stem cell-derived cardiomyocytes (hCMs and iCMs) in host myocardium. Methods and Results: ZFN technology was used to integrate a triple fusion RG, containing firefly luciferase (BLI), red fluorescent protein (RFP) and herpes simplex virus thymidine kinase (PET), under regulation of a constitutive ubiquitin promoter, into the AAVS1 locus of hESCs and iPSCs. hESCs and iPSCs monoclonal lines were then differentiated into hCMs and iCMs, respectively, under chemically defined conditions using CHIR and IWR small molecules to modulate Wnt pathway activity. Co-localization of cardiac phenotype and striated morphology was readily observed (Fig. A). Site-specific integration was confirmed by southern blot analysis. RG expression by BLI was maintained successfully over 3 months in vitro. The hCMs (n=6) and iCMs (n=1) were transplanted into murine myocardial injury model. Four weeks post-injury, LVEF was better preserved in all cell-treated vs. PBS-treated mice (26.50 ± 2.325 (N=6) vs. 15.40 ± 4.600 (N=3), p<0.05). Robust in vivo expression of ZFN-edited BLI and PET signal of engraftment persisted for 4-weeks duration (Figs. B and C). Conclusion: This study demonstrates the successful use of ZFN-edited cells to confirm the engraftment of hCMs and iCMs. ZFN technology enables targeted site-specific insertion of RGs to allow clinical translation of molecular imaging in cell therapy.

Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.

Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

ABSTRACTToxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity.

Site-specific insertion of selenium into the redox-active disulfide of the flavoprotein augmenter of liver regeneration

Augmenter of liver regeneration (sfALR) is a small disulfide-bridged homodimeric flavoprotein with sulfhydryl oxidase activity. Here, we investigate the catalytic and spectroscopic consequences of selectively replacing C145 by a selenocysteine to complement earlier studies in which random substitution of ∼90% of the 6 cysteine residues per sfALR monomer was achieved growing Escherichia coli on selenite. A selenocysteine insertion sequence (SECIS) element was installed within the gene for human sfALR. SecALR2 showed a spectrum comparable to that of wild-type sfALR. The catalytic efficiency of SecALR2 towards dithiothreitol was 6.8-fold lower than a corresponding construct in which position 145 was returned to a cysteine residue while retaining the additional mutations introduced with the SECIS element. This all-cysteine control enzyme formed a mixed disulfide between C142 and β-mercaptoethanol releasing C145 to form a thiolate-flavin charge transfer absorbance band at ∼530nm. In contrast, SecALR2 showed a prominent long-wavelength absorbance at 585nm consistent with the expectation that a selenolate would be a better charge-transfer donor to the isoalloxazine ring. These data show the robustness of the ALR protein fold towards the multiple mutations required to insert the SECIS element and provide the first example of a selenolate to flavin charge-transfer complex.

A novel transchromosomic system: stable maintenance of an engineered Mb-sized human genomic fragment translocated to a mouse chromosome terminal region

Transchromosomic (Tc) technology using human chromosome fragments (hCFs), or human artificial chromosomes (HACs), has been used for generating mice containing Mb-sized segments of the human genome. The most significant problem with freely segregating chromosomes with human centromeres has been mosaicism, possibly due to the instability of hCFs or HACs in mice. We report a system for the stable maintenance of Mb-sized human chromosomal fragments following translocation to mouse chromosome 10 (mChr.10). The approach utilizes microcell-mediated chromosome transfer and a combination of site-specific loxP insertion, telomere-directed chromosome truncation, and precise reciprocal translocation for the generation of Tc mice. Human chromosome 21 (hChr.21) was modified with a loxP site and truncated in homologous recombination-proficient chicken DT40 cells. Following transfer to mouse embryonic stem cells harboring a loxP site at the distal region of mChr.10, a ~4Mb segment of hChr.21 was translocated to the distal region of mChr.10 by transient expression of Cre recombinase. The residual hChr.21/mChr.10ter fragment was reduced by antibiotic negative selection. Tc mice harboring the translocated ~4Mb fragment were generated by chimera formation and germ line transmission. The hChr.21-derived Mb fragment was maintained stably in tissues in vivo and expression profiles of genes on hChr.21 were consistent with those seen in humans. Thus, Tc technology that enables translocation of human chromosomal regions onto host mouse chromosomes will be useful for studying in vivo functions of the human genome, and generating humanized model mice.

Foldback intercoil DNA and the mechanism of DNA transposition.

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180° and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Advances in Genome Engineering Approaches

Targeted genome editing is essential for functional characterization of a gene of interest. Targeted gene inactivation via homologous recombination made it feasible to create gene knockout animal models to ascertain the physiological role of the target genes; however, lower efficiency of site specific insertion of the genetically modified construct through homologous recombination has limited a wider applicability of this approach. Development of targeted gene knockdown through RNA interferce (RNAi) offered a cost effective, high-throughput alternative to homologous recombination, however, RNAi-mediated gene knockdown is incomplete, produces experiment to experiment variation, and could provide only a temporary inhibition of the gene function. Development of genome engineering methodologies utilizing nucleases linked to the guide sequences targeting a gene of interest, such as Zinc Finger Nucleases (ZFN), Transcription Activator like Effector Nucleases (TALEN) and Clustered Palindromic Repeats (CRISPR), are quite encouraging. A brief overview of recent advances in genome engineering approaches is provided with their respective advantages and limitations.

Critical Roles of SK3 Calcium-Activated Potassium Channels in the Repolarization of Atrial Myocytes

Small conductance Ca2+-activated K+ channels (SK channels) have been first identified in central nervous system, where they aid in integrating changes in intracellular Ca2+ with the intrinsic excitability of neurons and affect the synaptic transmission and plasticity. We have demonstrated that three members of SK channel family (SK1, SK2 and SK3) are present in human and mouse cardiac myocytes and contribute significantly to the repolarization process in both mouse and human atria. Moreover, the three members can heteromultimerize to form functional channels. In this study, we directly tested the contribution of SK3 channels to the overall repolarization of atrial action potentials. We used a mouse model with site-specific insertion of a tetracycline-based genetic switch in the 5' untranslated region of the KCNN3 (SK3 channel) gene so that SK3 expression could be decreased by dietary doxycycline administration without interfering with the normal profile of SK3 expression. Whole-cell patch-clamp recording showed a significant shortening of the action potential duration mainly at 90% repolarization (APD90) in atrial myocytes from the homozygous SK3T/T animals. Conversely, treatment with dietary doxycycline results in a significant prolongation of APD90 in atrial myocytes from SK3T/T animals. We further demonstrated that the shortening of action potential durations in SK3 over-expression mice predisposes the animals to inducible atrial arrhythmias. In conclusion, SK3 contributes toward atrial action potential repolarization, suggesting the pivotal role of the SK channels in atrial myocytes.

Molecular characterization of putative virulence determinants in Burkholderia pseudomallei.

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

Site-Specific, Insertional Inactivation of incA in Chlamydia trachomatis Using a Group II Intron

Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. However, the field still remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron’s substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron™ for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR, Southern blotting, and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA− mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at a multiplicity of infection greater than one. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron, ability to select for mutants, and intron stability in the absence of selection makes this method a powerful addition to the growing chlamydial molecular toolbox.

Nucleases for genome editing in crops

Site-specific nucleases create DNA double stranded breaks which, during repair, can be used to introduce site-specific point mutations, insertions and deletions into the plant genome. Identification of novel nucleases and further characterization of known nucleases, including Zinc finger nucleases, TAL effector-like endonucleases, Meganucleases and CRISPR–Cas, have increased the range and level of precision of methods available for modifying plant genomes. Precision, site-specific modification of plant genomes, known as genome editing, is emerging as a powerful tool for the improvement of crop species. This review discusses the functional and structural details of these nucleases and their potential applications for the genetic modification of plants.

Critical roles of a small conductance Ca2+-activated K+ channel (SK3) in the repolarization process of atrial myocytes

Small conductance Ca(2+)-activated K(+) channels (K(Ca)2 or SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca(2+) (Ca(i)²⁺) with membrane potentials. We have recently reported the functional expression of SK channels in human and mouse cardiac myocytes. Additionally, we have found that the channel is highly expressed in atria compared with the ventricular myocytes. We demonstrated that human cardiac myocytes expressed all three members of SK channels (SK1, 2, and 3); moreover, the different members are capable of forming heteromultimers. Here, we directly tested the contribution of SK3 to the overall repolarization of atrial action potentials. We took advantage of a mouse model with site-specific insertion of a tetracycline-based genetic switch in the 5' untranslated region of the KCNN3 (SK3 channel) gene (SK3(T/T)). The gene-targeted animals overexpress the SK3 channel without interfering with the normal profile of SK3 expression. Whole-cell, patch-clamp techniques show a significant shortening of the action potential duration mainly at 90% repolarization (APD90) in atrial myocytes from the homozygous SK3(T/T) animals. Conversely, treatment with dietary doxycycline results in a significant prolongation of APD90 in atrial myocytes from SK3(T/T) animals. We further demonstrate that the shortening of APDs in SK3 overexpression mice predisposes the animals to inducible atrial arrhythmias. SK3 channel contributes importantly towards atrial action potential repolarization. Our data suggest the important role of the SK3 isoform in atrial myocytes.

General approach for introduction of various chemical labels in specific RNA locations based on insertion of amino linkers.

Introduction of reporter groups at designed RNA sites is a widely accepted approach to gain information about the molecular environment of RNAs in their complexes with other biopolymers formed during various cellular processes. A general approach to obtain RNAs bearing diverse reporter groups at designed locations is based on site-specific insertion of groups containing primary aliphatic amine functions (amino linkers) with their subsequent selective derivatization by appropriate chemicals. This article is a brief review on methods for site-specific introduction of amino linkers in different RNAs. These methods comprise: (i) incorporation of a nucleoside carrying an amino-linker or a function that can be substituted with it into oligoribonucleotides in the course of their chemical synthesis; (ii) assembly of amino linker-containing RNAs from short synthetic fragments via their ligation; (iii) synthesis of amino linker-modified RNAs using T7 RNA polymerase; (iv) insertion of amino linkers into unmodified RNAs at functional groups of a certain type such as the 5'-phosphates and N7 of guanosine residues and (v) introduction of an amino linker into long highly structured RNAs exploiting an approach based on sequence-specific modification of nucleic acids. Particular reporter groups used for derivatization of amino linker-containing RNAs together with types of RNA derivatives obtained and fields of their application are presented.

Targeted transgene insertion into the AAVS1 locus driven by baculoviral vector‐mediated zinc finger nuclease expression in human‐induced pluripotent stem cells

The AAVS1 locus is viewed as a 'safe harbor' for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos.

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrpy11 mutant embryos or cardia bifida phenotypes in fautm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.

Back Cover: Rewiring Translation for Elongation Factor Tu-Dependent Selenocysteine Incorporation (Angew. Chem. Int. Ed. 5/2013)

Synthetic tRNA for selenoprotein production is described by D. Söll et al. in their Communication on page 1441 ff. The tRNA is a substrate for three E. coli proteins: seryl-tRNA synthetase (SerRS), selenocysteine synthase (SelA) generating Sec-tRNAUTu, and EF-Tu for Sec-tRNAUTu transport to the ribosome, which allows site-specific Sec insertion into proteins. This system has general utility in protein engineering, molecular biology, and disease research.

Dual Roles of Pseudomonas aeruginosa AlgE in Secretion of the Virulence Factor Alginate and Formation of the Secretion Complex

AlgE is a monomeric 18-stranded β-barrel protein required for secretion of the extracellular polysaccharide alginate in Pseudomonas aeruginosa. To assess the molecular mechanism of alginate secretion, AlgE was subjected to site-specific and FLAG epitope insertion mutagenesis. Except for β-strands 6 and 10, epitope insertions into the transmembrane β-strands abolished localization of AlgE to the outer membrane. Interestingly, an epitope insertion into β-strand 10 produced alginate and was only detectable in outer membranes isolated from cells grown on solid media. The deletion of nine C-terminal amino acid residues destabilized AlgE. Replacement of amino acids that constitute the highly electropositive pore constriction showed that individual amino acid residues have a specific function in alginate secretion. Two of the triple mutants (K47E+R353A+R459E and R74E+R362A+R459E) severely reduced alginate production. Mutual stability analysis using the algE deletion mutant PDO300ΔalgE(miniCTX) showed the periplasmic alginate biosynthesis proteins AlgK and AlgX were completely destabilized, while the copy number of the inner membrane c-di-GMP receptor Alg44 was reduced. Chromosomal integration of algE restored AlgK, AlgX, and Alg44, providing evidence for a multiprotein complex that spans the cell envelope. Periplasmic turn 4 of AlgE was identified as an important region for maintaining the stability of the putative multiprotein complex.