Escherichia coli Cytoplasmic Expression of Disulfide-Bonded Proteins: Side-by-Side Comparison between Two Competing Strategies.

The production of disulfide bond-containing recombinant proteins in Escherichia coli has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of E. coli: i) engineered strains with deletions in the disulfide reduction pathways, e.g. SHuffle, and ii) the co-expression of oxidative folding catalysts, e.g. CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.

Development of efficient modules for recombinant protein expression and periplasmic localisation in Pseudomonas bharatica CSV86T.

Escherichia coli has been widely employed as a host for heterologous protein expression. However, due to certain limitations, alternative hosts like Pseudomonas, Lactococcus and Bacillus are being explored. Pseudomonas bharatica CSV86T, a novel soil isolate, preferentially degrades wide range of aromatics over simple carbon sources like glucose and glycerol. Strain also possesses advantageous eco-physiological traits, making it an ideal host for engineering xenobiotic degradation pathways, which necessitates the development of heterologous expression systems. Based on the efficient growth, short lag-phase and rapid metabolism of naphthalene, Pnah and Psal promoters (regulated by NahR) were selected for expression. Pnah was found to be strong and leaky as compared to Psal, using 1-naphthol 2-hydroxylase (1NH, ∼66 kDa) as reporter gene in strain CSV86T. The Carbaryl hydrolase (CH, ∼72 kDa) from Pseudomonas sp. C5pp was expressed under Pnah in strain CSV86T and could successfully be translocated to the periplasm due to the presence of the Tmd + Sp sequence. The recombinant CH was purified from the periplasmic fraction and the kinetic characteristics were found to be similar to the native protein from strain C5pp. These results potentiate the suitability of P. bharatica CSV86T as a desirable host, while Pnah and the Tmd + Sp can be employed for overexpression and periplasmic localisation, respectively. Such tools find application in heterologous protein expression and metabolic engineering applications.

Specific N-terminal amino acids potentiate the periplasmic expression of single-chain variable fragments in Escherichia coli.

Single-chain variable fragments (ScFvs) are important in therapy, diagnosis and research because of their elevated antigen affinity and low immunogenicity. At present, high-yield scFv expression in Escherichia coli is limited by insoluble aggregation in the reducing environment of the cytoplasm or low yields in the periplasm. Here we achieved increased expression of scFvs in the periplasm by inserting optimal amino acids between the signal peptide and scFv. We constructed an expression library with three random amino acids at the scFv N-terminus, screened this library with a single-step colony assay and identified the specific sequences that boosted periplasmic expression of scFvs.

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway.

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of ß-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICBGlc, and then captured by ß-glucosidase.

The structural basis for high-affinity uptake of lignin-derived aromatic compounds by proteobacterial TRAP transporters.

The organic polymer lignin is a component of plant cell walls, which like (hemi)-cellulose is highly abundant in nature and relatively resistant to degradation. However, extracellular enzymes released by natural microbial consortia can cleave the ß-aryl ether linkages in lignin, releasing monoaromatic phenylpropanoids that can be further catabolised by diverse species of bacteria. Biodegradation of lignin is therefore important in global carbon cycling, and its natural abundance also makes it an attractive biotechnological feedstock for the industrial production of commodity chemicals. Whilst the pathways for degradation of lignin-derived aromatics have been extensively characterised, much less is understood about how they are recognised and taken up from the environment. The purple phototrophic bacterium Rhodopseudomonas palustris can grow on a range of phenylpropanoid monomers and is a model organism for studying their uptake and breakdown. R. palustris encodes a tripartite ATP-independent periplasmic (TRAP) transporter (TarPQM) linked to genes encoding phenylpropanoid-degrading enzymes. The periplasmic solute-binding protein component of this transporter, TarP, has previously been shown to bind aromatic substrates. Here, we determine the high-resolution crystal structure of TarP from R. palustris as well as the structures of homologous proteins from the salt marsh bacterium Sagittula stellata and the halophile Chromohalobacter salexigens, which also grow on lignin-derived aromatics. In combination with tryptophan fluorescence ligand-binding assays, our ligand-bound co-crystal structures reveal the molecular basis for high-affinity recognition of phenylpropanoids by these TRAP transporters, which have potential for improving uptake of these compounds for biotechnological transformations of lignin.

High-yield expression of periplasmic single-chain variable fragments by solid Escherichia coli cultures.

High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in Escherichia coli using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in E. coli cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated E. coli. This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.

Engineering a Supersecreting Strain of Escherichia coli by Directed Coevolution of the Multiprotein Tat Translocation Machinery.

Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed coevolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three supersecreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than those observed for wild-type TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of more efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis.

Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.

Disulfide-compatible phage-assisted continuous evolution in the periplasmic space.

The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.

Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

Conserved features of the MlaD domain aid the trafficking of hydrophobic molecules.

In Gram-negative bacteria, the maintenance of lipid asymmetry (Mla) system is involved in the transport of phospholipids between the inner (IM) and outer membrane. The Mla system utilizes a unique IM-associated periplasmic solute-binding protein, MlaD, which possesses a conserved domain, MlaD domain. While proteins carrying the MlaD domain are known to be primarily involved in the trafficking of hydrophobic molecules, not much is known about this domain itself. Thus, in this study, the characterization of the MlaD domain employing bioinformatics analysis is reported. The profiling of the MlaD domain of different architectures reveals the abundance of glycine and hydrophobic residues and the lack of cysteine residues. The domain possesses a conserved N-terminal region and a well-preserved glycine residue that constitutes a consensus motif across different architectures. Phylogenetic analysis shows that the MlaD domain archetypes are evolutionarily closer and marked by the conservation of a functionally crucial pore loop located at the C-terminal region. The study also establishes the critical role of the domain-associated permeases and the driving forces governing the transport of hydrophobic molecules. This sheds sufficient light on the structure-function-evolutionary relationship of MlaD domain. The hexameric interface analysis reveals that the MlaD domain itself is not a sole player in the oligomerization of the proteins. Further, an operonic and interactome map analysis reveals that the Mla and the Mce systems are dependent on the structural homologs of the nuclear transport factor 2 superfamily.

Improving the yield of recalcitrant Nanobodies® by simple modifications to the standard protocol.

Nanobodies are single-domain antibody constructs derived from the variable regions of heavy chain only (VHH) camelid IgGs. Their small size and single gene format make them amenable to various molecular biology applications that require a protein affinity-based approach. These features, in addition to their high solubility, allows their periplasmic expression, extraction and purification in E. coli systems with relative ease, using standardized protocols. However, some Nanobodies are recalcitrant to periplasmic expression, extraction and purification within E. coli systems. To improve their expression would require either a change in the expression host, vector or an increased scale of expression, all of which entail an increase in the complexity of their expression, and production cost. However, as shown here, specific changes in the existing standard E. coli culture protocol, aimed at reducing breakdown of selective antibiotic pressure, increasing the initial culture inoculum and improving transport to the periplasmic space, rescued the expression of several such refractory Nanobodies. The periplasmic extraction protocol was also changed to ensure efficient osmolysis, prevent both protein degradation and prevent downstream chelation of Ni2+ ions during IMAC purification. Adoption of this protocol will lead to an improvement of the expression of Nanobodies in general, and specifically, those that are recalcitrant.

The periplasmic component of the DctPQM TRAP-transporter is part of the DctS/DctR sensory pathway in Rhodobacter sphaeroides.

Rhodobacter sphaeroides can use C4-dicarboxylic acids to grow heterotrophically or photoheterotropically, and it was previously demonstrated in Rhodobacter capsulatus that the DctPQM transporter system is essential to support growth using these organic acids under heterotrophic but not under photoheterotrophic conditions. In this work we show that in R. sphaeroides this transporter system is essential for photoheterotrophic and heterotrophic growth, when C4-dicarboxylic acids are used as a carbon source. We also found that over-expression of dctPQM is detrimental for photoheterotrophic growth in the presence of succinic acid in the culture medium. In agreement with this, we observed a reduction of the dctPQM promoter activity in cells growing under these conditions, indicating that the amount of DctPQM needs to be reduced under photoheterotrophic growth. It has been reported that the two-component system DctS and DctR activates the expression of dctPQM. Our results demonstrate that in the absence of DctR, dctPQM is still expressed albeit at a low level. In this work, we have found that the periplasmic component of the transporter system, DctP, has a role in both transport and in signalling the DctS/DctR two-component system.

Genome-scale mutant fitness reveals versatile c-type cytochromes in Shewanella oneidensis MR-1.

Shewanella has been widely investigated for its metabolic versatility and use of a large number of extracellular electron acceptors. Many c-type cytochromes are responsible for this diversity, mainly in condition-specific fashions. By using genome-scale mutant fitness data, we studied which genes (particularly c-type cytochromes) were used to coordinate various electron transfer processes in the present work. First, by integrating fitness profiles with protein-protein interaction (PPI) networks, we showed that the genes with a high total fitness value were generally more important in PPI networks than those with low fitness values. Then, we identified genes that are important across many experiments, and further fitness analysis confirmed five versatile c-type cytochromes: ScyA (SO0264), PetC (SO0610), CcoP (SO2361), CcoO (SO2363) and CytcB (SO4666), which are considered to be crucial in most experimental conditions. Finally, we demonstrated a mediating role in the periplasm for the less-reported CytcB by combining protein structure, subcellular localization and disordered region analysis. Comparative genome analysis further revealed that it is distinctive in Shewanella species. Collectively, these results suggest that periplasmic electron transfer processes are more diverse and flexible than previously reported, giving insight for further experimental studies of Shewanella oneidensis MR-1.

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases.

The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway. The diversity of the family is revealed by the generation and analysis of a sequence similarity network for the superfamily, with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. Three common fusions were identified: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. Additionally, unexpected fusions of the monoPGT with members of the polytopic PGT superfamily were discovered, implying a possible evolutionary link through the shared polyprenol phosphate substrate. Notably, a phylogenetic reconstruction of the monoPGT superfamily shows a radial burst of functionalization, with a minority of members comprising only the minimal PGT catalytic domain. The commonality and identity of the fusion partners in the monoPGT superfamily is consistent with advantageous colocalization of pathway members at membrane interfaces.

A mutagenic screen reveals NspS residues important for regulation of Vibrio cholerae biofilm formation.

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.

Polarity/charge as a determinant of translocase requirements for membrane protein insertion.

The YidC insertase of Escherichia coli inserts membrane proteins with small periplasmic loops (~20 residues). However, it has difficulty transporting loops that contain positively charged residues compared to negatively charged residues and, as a result, increasing the positive charge has an increased requirement for the Sec machinery as compared to negatively charged loops (Zhu et al., 2013; Soman et al., 2014). This suggested that the polarity and charge of the periplasmic regions of membrane proteins determine the YidC and Sec translocase requirements for insertion. Here we tested this polarity/charge hypothesis by showing that insertion of our model substrate protein procoat-Lep can become YidC/Sec dependent when the periplasmic loop was converted to highly polar even in the absence of any charged residues. Moreover, adding a number of hydrophobic amino acids to a highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent membrane proteins. We also demonstrate that the length of the procoat-Lep loop is indeed a determinant for Sec-dependence by inserting alanine residues that do not markedly change the overall hydrophilicity of the periplasmic loop. Taken together, the results support the polarity/charge hypothesis as a determinant for the translocase requirement for procoat insertion.

Method for efficient soluble expression and purification of recombinant human interleukin-15.

Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The target construct periplasmic expression was evaluated in four strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta 2 (DE3) and Rosetta-gami 2 (DE3). Soluble periplasmic expression of IL-15 was highest in Rosetta-gami 2 (DE3) followed by Rossetta 2 (DE3) whereas negligible expression was observed with rest of two expression host. Best expression clone was selected for purification by dye ligand affinity chromatography. Purified rhIL-15 was characterized by SDS-PAGE, Western blotting and SEC-HPLC. This is the first report of functional recombinant human interleukin-15 being expressed and purified with yield of 120 mg/L in the periplasmic space of E. coli.

Crystal structure of NirF: insights into its role in heme d1 biosynthesis.

Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa can respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes, for example, to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infections. A central step in denitrification is the reduction of nitrite to nitric oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme d1 . While heme d1 biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydroheme d1 , the last intermediate of heme d1 biosynthesis. We found that NirF forms a bottom-to-bottom ß-propeller homodimer and confirmed this by multi-angle light and small-angle X-ray scattering. The N termini are adjacent to each other and project away from the core structure, which hints at simultaneous membrane anchoring via both N termini. Further, the complex with dihydroheme d1 allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a ΔnirF strain of P. aeruginosa. Together, these data suggest that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d1 derivatives. DATABASE: Structural data are available in PDB database under the accession numbers 6TV2 and 6TV9.

A structure-function study of ZraP and ZraS provides new insights into the two-component system Zra.

BACKGROUND: Zra belongs to the envelope stress response (ESR) two-component systems (TCS). It is atypical because of its third periplasmic repressor partner (ZraP), in addition to its histidine kinase sensor protein (ZraS) and its response regulator (ZraR) components. Furthermore, although it is activated by Zn2+, it is not involved in zinc homeostasis or protection against zinc toxicity. Here, we mainly focus on ZraS but also provide information on ZraP. METHODS: The purified periplasmic domain of ZraS and ZraP were characterized using biophysical and biochemical technics: multi-angle laser light scattering (MALLS), circular dichroism (CD), differential scanning fluorescence (DSF), inductively coupled plasma atomic emission spectroscopy (ICP-AES), cross-linking and small-angle X-ray scattering (SAXS). In-vivo experiments were carried out to determine the redox state of the cysteine residue in ZraP and the consequences for the cell of an over-activation of the Zra system. RESULTS: We show that ZraS binds one Zn2+ molecule with high affinity resulting in conformational changes of the periplasmic domain, consistent with a triggering function of the metal ion. We also demonstrate that, in the periplasm, the only cysteine residue of ZraP is at least partially reduced. Using SAXS, we conclude that the previously determined X-ray structure is different from the structure in solution. CONCLUSION: Our results allow us to propose a general mechanism for the Zra system activation and to compare it to the homologous Cpx system. GENERAL SIGNIFICANCE: We bring new input on the so far poorly described Zra system and notably on ZraS.