Divergent synthesis and biological evaluation of 2-(trifluoromethyl)pyridines as virulence-attenuating inverse agonists targeting PqsR.

A short and divergent route towards new derivatives of 2-(trifluoromethyl)pyridines as potent inverse agonists of the bacterial target PqsR against Pseudomonas aeruginosa (PA) infections is described. This Gram-negative pathogen causes severe nosocomial infections and common antibiotic treatment options are rendered ineffective due to resistance issues. Based on an earlier identified optimized hit, we conducted derivatization and rigidification attempts employing two central building blocks. The western part of the molecule is built up via a 2-(trifluoromethyl)pyridine head group equipped with a terminal alkyne. The eastern section is then introduced through aryliode motifs exploiting Sonogashira as well as Suzuki-type chemistry. Subsequent modification provided quick access to an array of compounds, allowed for deep SAR insights, and enabled to optimize the hit scaffold into a lead structure of nanomolar potency combined with favorable in vitro ADME/T features.

A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) - a crucial transcriptional regulator serving major functions in PA virulence - can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10-9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development.

Measuring protein insertion areas in lipid monolayers by fluorescence correlation spectroscopy.

Membrane insertion of protein domains is an important step in many membrane remodeling processes, for example, in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. To our knowledge, this is the first optical measurement of this quantity on a system in equilibrium with direct determination of the number of inserted protein and no further assumptions concerning the binding thermodynamics. Whereas macroscopic total area changes in lipid monolayers are typically measured on a Langmuir film balance, finding the number of inserted proteins without perturbing the system and quantitating any small area changes has posed a challenge. Here, we address both issues by performing two-color fluorescence correlation spectroscopy directly on the monolayer. With a fraction of the protein being fluorescently labeled, the number of inserted proteins is determined in situ without resorting to invasive techniques such as collecting the monolayer by aspiration. The second color channel is exploited to monitor a small fraction of labeled lipids to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm2 was obtained for Sar1 in monolayers from a lipid mixture typically used in COPII reconstitution experiments, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to also be applied to laterally heterogeneous monolayers and bilayers.

RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.

The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites.

Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).

Structure of a glutamine donor mimicking inhibitory peptide shaped by the catalytic cleft of microbial transglutaminase.

The protein cross-linking enzyme transglutaminase from Streptomyces mobaraensis (MTG) is frequently used to modify therapeutic proteins. In order to reveal the binding mode of glutamine donor substrates, we have now crystallized MTG covalently linked to large inhibitory peptides. A series of peptide structures were examined but DIPIGSKMTG, which was chloroacetylated at serine, was the only inhibitory molecule that resulted in an interpretable density map. We found that, besides the warhead (modified Ser6), Ile4 and Gly5 of the inhibitory peptide occupy the tight but extended hydrophobic bottom of the MTG-binding cleft. Both termini of the peptide protrude along the cleft walls almost perpendicular to the bottom of the extended cleft. This peptide model suggests a zipper-like cross-linking mechanism of self-assembled substrate proteins by MTG.

Destructive twisting of neutral metalloproteases: the catalysis mechanism of the Dispase autolysis-inducing protein from Streptomyces mobaraensis DSM 40487.

The Dispase autolysis-inducing protein (DAIP) is produced by Streptomyces mobaraensis to disarm neutral metalloproteases by decomposition. The absence of a catalytic protease domain led to the assumption that the seven-bladed ß-propeller protein DAIP causes structural modifications, thereby triggering autolysis. Determination of protein complexes consisting of DAIP and thermolysin or DAIP and a nonfunctional E138A bacillolysin variant supported this postulation. Protein twisting was indicated by DAIP-mediated inhibition of thermolysin while bacillolysin underwent immediate autolysis under the same conditions. Interestingly, an increase in SYPRO orange fluorescence allowed tracking of the fast degradation process. Similarly rapid autolysis of thermolysin mediated by DAIP was only observed upon the addition of amphiphilic compounds, which probably amplify the induced structural changes. DAIP further caused degradation of FITC-labeled E138A bacillolysin by trypsin, as monitored by a linear decrease in fluorescence polarization. The kinetic model, calculated from the obtained data, suggested a three-step mechanism defined by (a) fast DAIP-metalloprotease complex formation, (b) slower DAIP-mediated protein twisting, and (c) fragmentation. These results were substantiated by crystallized DAIP attached to a C-terminal helix fragment of thermolysin. Structural superposition of the complex with thermolysin is indicative of a conformational change upon binding to DAIP. Importantly, the majority of metalloproteases, also including homologs from various pathogens, are highly conserved at the autolysis-prone peptide bonds, suggesting their susceptibility to DAIP-mediated decomposition, which may offer opportunities for pharmaceutical applications. DATABASES: The atomic coordinates and structure factors (PDB ID: 6FHP) have been deposited in the Protein Data Bank (http://www.pdb.org/). ENZYMES: Aureolysin, EC 3.4.24.29; bacillolysin (Dispase, Gentlyase), EC 3.4.24.28; lasB (elastase), EC 3.4.24.4; subtilisin, EC 3.4.21.62; thermolysin, EC 3.4.24.27; transglutaminase, EC 2.3.2.13; trypsin, EC 3.4.21.4; vibriolysin (hemagglutinin(HA)/protease), EC 3.4.24.25.

Design, development and application of whole-cell based antibiotic-specific biosensor.

Synthetic biology techniques hold great promise for optimising the production of natural products by microorganisms. However, evaluating the phenotype of a modified bacterium represents a major bottleneck to the engineering cycle - particularly for antibiotic-producing actinobacteria strains, which grow slowly and are challenging to genetically manipulate. Here, we report the generation and application of antibiotic-specific whole-cell biosensor derived from TetR transcriptional repressor for use in identifying and optimising antibiotic producers. The constructed biosensor was successfully used to improve production of polyketide antibiotic pamamycin. However, an initial biosensor based on native genetic elements had inadequate dynamic and operating ranges. To overcome these limitations, we fine-tuned biosensor performance through alterations of the promoter and operator of output module and the ligand affinity of transcription factor module, which enabled us to deduce recommendations for building and application of actinobacterial biosensors.

The invasin D protein from Yersinia pseudotuberculosis selectively binds the Fab region of host antibodies and affects colonization of the intestine.

Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. The Y. pseudotuberculosis invasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37 °C and expressed in vivo 2-4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical ß-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host.

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis.

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPIp ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPIp exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPIp accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPIp for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications.

Yersinia Pseudotuberculosis Modulates Regulatory T Cell Stability via Injection of Yersinia Outer Proteins in a Type III Secretion System-Dependent Manner.

Adaptive immunity is essentially required to control acute infection with enteropathogenic Yersinia pseudotuberculosis (Yptb). We have recently demonstrated that Yptb can directly modulate naïve CD4+ T cell differentiation. However, whether fully differentiated forkhead box protein P3 (Foxp3+) regulatory T cells (Tregs), fundamental key players to maintain immune homeostasis, are targeted by Yptb remains elusive. Here, we demonstrate that within the CD4+ T cell compartment Yptb preferentially targets Tregs and injects Yersinia outer proteins (Yops) in a process that depends on the type III secretion system and invasins. Remarkably, Yop-translocation into ex vivo isolated Foxp3+ Tregs resulted in a substantial downregulation of Foxp3 expression and a decreased capacity to express the immunosuppressive cytokine interleukin-10 (IL-10). Together, these findings highlight that invasins are critically required to mediate Yptb attachment to Foxp3+ Tregs, which allows efficient Yop-translocation and finally enables the modulation of the Foxp3+ Tregs' suppressive phenotype.

Identification, biochemical characterization and crystallization of the central region of human ATG16L1.

ATG16L1 plays a major role in autophagy. It acts as a molecular scaffold which mediates protein-protein interactions essential for autophagosome formation. The ATG12~ATG5-ATG16L1 complex is one of the key complexes involved in autophagosome formation. Human ATG16L1 comprises 607 amino acids with three functional domains named ATG5BD, CCD and WD40, where the C-terminal WD40 domain represents approximately 50% of the full-length protein. Previously, structures of the C-terminal WD40 domain of human ATG16L1 as well as of human ATG12~ATG5 in complex with the ATG5BD of ATG16L1 have been reported. However, apart from the ATG5BD, no structural information for the N-terminal half, including the CCD, of human ATG16L1 is available. In this study, the authors aimed to structurally characterize the N-terminal half of ATG16L1. ATG16L111-307 in complex with ATG5 has been purified and crystallized in two crystal forms. However, both crystal structures revealed degradation of ATG16L1, resulting in crystals comprising only full-length ATG5 and the ATG5BD of ATG16L1. The structures of ATG5-ATG5BD in two novel crystal forms are presented, further supporting the previously observed dimerization of ATG5-ATG16L1. The reported degradation points towards a high instability at the linker region between the ATG5BD and the CCD in ATG16L1. Based on this observation and further biochemical analysis of ATG16L1, a stable 236-amino-acid subfragment comprising residues 72-307 of the N-terminal half of ATG16L1, covering the residual, so far structurally uncharacterized region of human ATG16L1, was identified. Here, the identification, purification, biochemical characterization and crystallization of the proteolytically stable ATG16L172-307 subfragment are reported.

UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene.

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.

Structure of the WD40-domain of human ATG16L1.

Autophagy-related protein ATG16L1 is a component of the mammalian ATG12∼ATG5/ATG16L1 complex, which acts as E3-ligase to catalyze lipidation of LC3 during autophagosome biogenesis. The N-terminal part of ATG16L1 comprises the ATG5-binding site and coiled-coil dimerization domain, both also present in yeast ATG16 and essential for bulk and starvation induced autophagy. While absent in yeast ATG16, mammalian ATG16L1 further contains a predicted C-terminal WD40-domain, which has been shown to be involved in mediating interaction with diverse factors in the context of alternative functions of autophagy, such as inflammatory control and xenophagy. In this work, we provide detailed information on the domain boundaries of the WD40-domain of human ATG16L1 and present its crystal structure at a resolution of 1.55 Å.

Structure of the Y. pseudotuberculosis adhesin InvasinE.

Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin-family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin-family of proteins. The sequence of the C-terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin-family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin-like domains. The structure reveals a rod shaped architecture, confirmed by small angle X-ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C-type lectin-like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C-type lectin-like domain in InvasinE lacks motifs required for Ca2+ /carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and ß1 -integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host-cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin-family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization.

Structural insights into the architecture of the Shigella flexneri virulence factor IcsA/VirG and motifs involved in polar distribution and secretion.

IcsA/VirG is a key virulence factor of the human pathogen Shigella flexneri, acting as both an adhesin and actin-polymerizing factor during infection. We identified a soluble expression construct of the IcsA/VirG α-domain using the ESPRIT library screening system and determined its structure to 1.9Å resolution. In addition to the previously characterized autochaperone domain, our structure reveals a new domain, which shares a common fold with the autochaperone domains of various autotransporters. We further provide insight into the previously structurally uncharacterized ß-helix domain that harbors the polar targeting motif and passenger-associated transport repeat. This structure is the first of any member of the recently identified passenger-associated transport repeat-containing autotransporters. Thus, it provides new insights into the overall architecture of this class of autotransporters, the function of the identified additional autochaperone domain and the structural properties of motifs involved in polar targeting and secretion of the Shigella flexneri virulence factor IcsA/VirG.

The AibR-isovaleryl coenzyme A regulator and its DNA binding site - a model for the regulation of alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus.

Isovaleryl coenzyme A (IV-CoA) is an important building block of iso-fatty acids. In myxobacteria, IV-CoA is essential for the formation of signaling molecules involved in fruiting body formation. Leucine degradation is the common source of IV-CoA, but a second, de novo biosynthetic route to IV-CoA termed AIB (alternative IV-CoA biosynthesis) was recently discovered in M. xanthus. The AIB-operon contains the TetR-like transcriptional regulator AibR, which we characterize in this study. We demonstrate that IV-CoA binds AibR with micromolar affinity and show by gelshift experiments that AibR interacts with the promoter region of the AIB-operon once IV-CoA is present. We identify an 18-bp near-perfect palindromic repeat as containing the AibR operator and provide evidence that AibR also controls an additional genomic locus coding for a putative acetyl-CoA acetyltransferase. To elucidate atomic details, we determined crystal structures of AibR in the apo, the IV-CoA- and the IV-CoA-DNA-bound state to 1.7 Å, 2.35 Å and 2.92 Å, respectively. IV-CoA induces partial unfolding of an α-helix, which allows sequence-specific interactions between AibR and its operator. This study provides insights into AibR-mediated regulation and shows that AibR functions in an unusual TetR-like manner by blocking transcription not in the ligand-free but in the effector-bound state.

Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed ß-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

RflM mediates target specificity of the RcsCDB phosphorelay system for transcriptional repression of flagellar synthesis in Salmonella enterica.

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post-transcriptional levels. The flagellar master regulator FlhD4 C2 resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD4 C2 -dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM-dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis.

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.

Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor-, and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid-based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975-1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.