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Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm. [ABSTRACT FROM AUTHOR]

Typhoidal Salmonella enterica serovars, such as Typhi and Paratyphi A, cause severe systemic infections, thereby posing a significant threat as human-adapted pathogens. This study focuses on cytolysin A (ClyA), a virulence factor essential for bacterial dissemination within the human body. We show that ClyA is exclusively expressed by intracellular S. Paratyphi A within the Salmonella-containing vacuole (SCV), regulated by the PhoP/Q system and SlyA. ClyA localizes in the bacterial periplasm, suggesting potential secretion. Deletion of TtsA, an essential Type 10 Secretion System component, completely abolishes intracellular ClyA detection and its presence in host cell supernatants. Host cells infected with wild-type S. Paratyphi A contain substantial ClyA, with supernatants capable of lysing neighboring cells. Notably, ClyA selectively lyses macrophages and erythrocytes while sparing epithelial cells. These findings identify ClyA as an intracellularly induced cytolysin, dependent on the SCV environment and secreted via a Type 10 Secretion System, with specific cytolytic activity. Typhoidal Salmonella enterica strains cause serious infections in humans. Here, Krone et al show that the bacterial protein cytolysin A is secreted intracellularly where it selectively kills specific cell types and helps to establish bacterial infection. [ABSTRACT FROM AUTHOR]

This review strives to assemble a set of molecular design principles that enables the delivery of antibiotic warheads to Gram-negative bacterial targets (ESKAPE pathogens) using iron-chelating siderophores, known as the Trojan Horse strategy for antibiotic development. Principles are derived along two main lines. First, archetypical siderophores and their conjugates are used as case studies for native iron transport. They enable the consideration of the correspondence of iron transport and antibacterial target location. The second line of study charts the rationale behind the clinical antibiotic cefiderocol. It illustrates the potential versatility for the design of new Trojan Horse-based antibiotics. Themes such as matching the warhead to a location where the siderophore delivers its cargo (i.e., periplasm vs. cytoplasm), whether or not a cleavable linker is required, and the relevance of cheaters to the effectiveness and selectivity of new conjugates will be explored. The effort to articulate rules has identified gaps in the current understanding of iron transport pathways and suggests directions for new investigations. [ABSTRACT FROM AUTHOR]

Under standard laboratory conditions, Synechococcus elongatus PCC 7942 lacks EcaASyn, a periplasmic carbonic anhydrase (CA). In this study, a S. elongatus transformant was created that expressed the homologous EcaACya from Cyanothece sp. ATCC 51142. This additional external CA had no discernible effect on the adaptive responses and physiology of cells exposed to changes similar to those found in S. elongatus natural habitats, such as fluctuating CO2 and HCO3− concentrations and ratios, oxidative or light stress, and high CO2. The transformant had a disadvantage over wild-type cells under certain conditions (Na+ depletion, a reduction in CO2). S. elongatus cells lacked their own EcaASyn in all experimental conditions. The results suggest the presence in S. elongatus of mechanisms that limit the appearance of EcaASyn in the periplasm. For the first time, we offer data on the expression pattern of CCM-associated genes during S. elongatus adaptation to CO2 replacement with HCO3−, as well as cell transfer to high CO2 levels (up to 100%). An increase in CO2 concentration coincides with the suppression of the NDH-14 system, which was previously thought to function constitutively. [ABSTRACT FROM AUTHOR]

The flagellar motor protein FliL is conserved across many microbes, but its exact role has been obscured by varying fliL mutant phenotypes. We reanalyzed results from fliL studies and found they utilized alleles that differed in the amount of N- and C-terminal regions that were retained. Alleles that retain the N-terminal cytoplasmic and transmembrane helix (TM) regions in the absence of the C-terminal periplasmic domain result in loss of motility, while alleles that completely lack the N-terminal region, independent of the periplasmic domain, retain motility. We then tested this prediction in Helicobacter pylori fliL and found support for the idea. This analysis suggests that FliL function may be more conserved across bacteria than previously thought, that it is not essential for motility, and that the N-terminal region has the negative ability to regulate motor function. IMPORTANCE FliL is a protein found in the flagellar motor of bacteria, but what it does was not clear. To study FliL function, scientists often remove it and see what happens. Loss of FliL was thought to have different effects depending on the microbe. We uncovered, however, that part of the confusion arose because scientists inadvertently removed different parts of the protein. Our analysis and data suggest that leaving the N-terminal regions blocks motility, while fully removing FliL allows normal motility. This finding will help scientists understand FliL because it clarifies what needs to be removed to fully eliminate the protein, and also that the N-terminal region can block motility.

Background: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation. Results: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1–84, somatostatin 1–28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6–2.6 g L−1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds. Conclusion: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis. [ABSTRACT FROM AUTHOR]

Abstract Background The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection phenotype. Results This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation. Conclusions These findings, coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining as well as providing insight into the binding dynamics of these minimal DNA ligases.

The gram-negative bacterium Bradyrhizobium japonicum can take up structurally dissimilar ferric siderophores from the environment (xenosiderophores) to meet its nutritional iron requirements. Siderophore-bound iron transported into the periplasm is reduced to the ferrous form by FsrB, dissociated from the siderophore and the free ion is then transported into the cytoplasm by the ferrous iron transporter FeoAB. Here, we identified the RND family exporter genes exsFG and exsH in a selection for secondary site suppressor mutants that restore growth of an fsrB mutant on the siderophores ferrichrome or ferrioxamine. The low level of radiolabel accumulation from 55Fe-labeled ferrichrome or ferrioxamine observed in the fsrB mutant was restored to wild type levels in the fsrB exsG mutant. Moreover, the exsG mutant accumulated more radiolabel from the 55Fe-labeled siderophores than the wild type, but radiolabel accumulation from inorganic 55Fe was similar in the two strains. Thus, ExsFGH exports siderophore-bound iron, but not inorganic iron. The rescued fsrB exsG mutant required feoB for growth, indicating that ExsFGH acts on those siderophores in the periplasm. The exsG mutant was more sensitive to the siderophore antibiotic albomycin than the wild type, whereas the fsrB mutant was more resistant. This suggests ExsFGH normally exports ferrated albomycin. B. japonicum is naturally resistant to many antibiotics. The exsG strain was very sensitive to tetracycline, but not to six other antibiotics tested. We conclude that ExsFGH is a broad substrate exporter that is needed to maintain siderophore homeostasis in the periplasm. [ABSTRACT FROM AUTHOR]

The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide bond formation, required by some proteins (such as antibody fragments) for correct folding and function. It also permits simpler protein release and downstream processing than cytoplasmic accumulation. As such, targeting of recombinant proteins to the E. coli periplasm is a key strategy in biologic manufacture. However, expression and translocation of each recombinant protein requires optimisation including selection of the best signal peptide and growth and production conditions. Traditional methods require separation and analysis of protein compositions of periplasmic and cytoplasmic fractions, a time- and labour-intensive method that is difficult to parallelise. Therefore, approaches for high throughput quantification of periplasmic protein accumulation offer advantages in rapid process development. ● Production of recombinant proteins in the E. coli periplasm is often advantageous. ● Approaches to screen periplasmic production lag behind those for the cytoplasm. ● A range of current approaches are reviewed and compared. [ABSTRACT FROM AUTHOR]

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.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host–bacteria interactions which may not be defined solely by protein–protein and protein–outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs. [ABSTRACT FROM AUTHOR]

Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract of E. coli to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads. We next designed conjugates consisting of (1) an N-terminal siderophore to facilitate uptake, (2) a protease-cleavable linker, and (3) an amine-containing antibiotic. Using this strategy, we converted daptomycin-which by itself is active only against Gram-positive bacteria-into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophore-facilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform's utility for development of protease-activated prodrugs, including Trojan horse antibiotics.

BACKGROUND:The reconstruction of metabolic networks and the three-dimensional coverage of protein structures have reached the genome-scale in the widely studied Escherichia coli K-12 MG1655 strain. The combination of the two leads to the formation of a structural systems biology framework, which we have used to analyze differences between the reactive oxygen species (ROS) sensitivity of the proteomes of sequenced strains of E. coli. As proteins are one of the main targets of oxidative damage, understanding how the genetic changes of different strains of a species relates to its oxidative environment can reveal hypotheses as to why these variations arise and suggest directions of future experimental work. RESULTS:Creating a reference structural proteome for E. coli allows us to comprehensively map genetic changes in 1764 different strains to their locations on 4118 3D protein structures. We use metabolic modeling to predict basal ROS production levels (ROStype) for 695 of these strains, finding that strains with both higher and lower basal levels tend to enrich their proteomes with antioxidative properties, and speculate as to why that is. We computationally assess a strain's sensitivity to an oxidative environment, based on known chemical mechanisms of oxidative damage to protein groups, defined by their localization and functionality. Two general groups - metalloproteins and periplasmic proteins - show enrichment of their antioxidative properties between the 695 strains with a predicted ROStype as well as 116 strains with an assigned pathotype. Specifically, proteins that a) utilize a molybdenum ion as a cofactor and b) are involved in the biogenesis of fimbriae show intriguing protective properties to resist oxidative damage. Overall, these findings indicate that a strain's sensitivity to oxidative damage can be elucidated from the structural proteome, though future experimental work is needed to validate our model assumptions and findings. CONCLUSION:We thus demonstrate that structural systems biology enables a proteome-wide, computational assessment of changes to atomic-level physicochemical properties and of oxidative damage mechanisms for multiple strains in a species. This integrative approach opens new avenues to study adaptation to a particular environment based on physiological properties predicted from sequence alone.

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.

The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 µg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE-optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro. [ABSTRACT FROM AUTHOR]

Copper avidly binds thiols and is redox active, and it follows that one element of copper toxicity may be the generation of undesirable disulfide bonds in proteins. In the present study, copper oxidized the model thiol N‐acetylcysteine in vitro. Alkaline phosphatase (AP) requires disulfide bonds for activity, and copper activated reduced AP both in vitro and when it was expressed in the periplasm of mutants lacking their native disulfide‐generating system. However, AP was not activated when it was expressed in the cytoplasm of copper‐overloaded cells. Similarly, this copper stress failed to activate OxyR, a transcription factor that responds to the creation of a disulfide bond. The elimination of cellular disulfide‐reducing systems did not change these results. Nevertheless, in these cells, the cytoplasmic copper concentration was high enough to impair growth and completely inactivate enzymes with solvent‐exposed [4Fe‐4S] clusters. Experiments with N‐acetylcysteine determined that the efficiency of thiol oxidation is limited by the sluggish pace at which oxygen regenerates copper(II) through oxidation of the thiyl radical–Cu(I) complex. We conclude that this slow step makes copper too inefficient a catalyst to create disulfide stress in the thiol‐rich cytoplasm, but it can still impact the few thiol‐containing proteins in the periplasm. It also ensures that copper accumulates intracellularly in the Cu(I) valence. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria can resist antibiotics by changing the permeability via their outer membrane. These bacteria have a complex cell envelope that incorporates an outer membrane separating the periplasm from the external environment. This outer membrane contains many protein channels, also known as porins or nanopores, which mainly allow the influx of hydrophilic compounds, including antibiotics. One probable way bacteria may possibly develop antibiotic resistance is by reworking to reduce the inflow through these outer membrane porins or nanopores. The challenge now is to recognize and potentially comprehend the molecular basis of permeability via the bacterial outer membrane. To address this challenge, this assessment builds upon the author's previous work to develop a comprehensive understanding of membrane porins and their crucial role in the influx of antibiotics and solutes. Furthermore, the work aspires to investigate the bacterial response to antibiotic membrane permeability and nurture discussion toward further exploration of the physicochemical parameters governing the translocation/transport of antibiotics through bacterial membrane porins. By augmenting our understanding of these mechanisms, we may devise novel approaches to mitigate antibiotic resistance in Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo “steady state” redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was almost fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the steady state periplasmic thiol-redox potential from −228 mV to a more reducing −243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

Two-component systems (TCSs) are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In Escherichia coli, the CusS-CusR TCS up-regulates the cusCFBA genes under increased periplasmic Cu(I)/Ag(I) concentrations to help maintain metal ion homeostasis. The CusS histidine kinase is a homodimeric integral membrane protein that binds to periplasmic Cu(I)/Ag(I) and transduces a signal to its cytoplasmic kinase domain. However, the mechanism of how metal binding in the periplasm activates autophosphorylation in the cytoplasm is unknown. Here, we report that only one of the two metal ion-binding sites in CusS enhances dimerization of the sensor domain. Utilizing nanodisc technology to study full-length CusS, we show that metal-induced dimerization in the sensor domain triggers kinase activity in the cytoplasmic domain. We also investigated autophosphorylation in the cytoplasmic domain of CusS and phosphotransfer between CusS and CusR. In vitro analyses show that CusS autophosphorylates its conserved H271 residue at the N1 position of the histidine imidazole. The phosphoryl group is removed by the response regulator CusR in a reaction that requires a conserved aspartate at position 51. Functional analyses in vivo of CusS and CusR variants with mutations in the autophosphorylation or phosphoacceptor residues suggest that the phosphotransfer event is essential for metal resistance in E. coli Biochemical analysis shows that the CusS dimer autophosphorylates using a cis mechanism. Our results support a signal transduction model in which rotation and bending movements in the cytoplasmic domain maintain the mode of autophosphorylation.

Abstract High‐value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via the Sec pathway as it, among other advantages, enables disulfide bond formation and simplifies downstream processing. However, the Sec system cannot transport complex or rapidly folding proteins, as it only transports proteins in an unfolded state. The Tat system also transports proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Most of the studies related to Tat secretion have used the well‐studied TorA signal peptide that is Tat‐specific, but this signal peptide also tends to induce degradation of the protein of interest, resulting in lower yields. This makes it difficult to use Tat in the industry. In this study, we show that a model disulfide bond‐containing protein, YebF, can be exported to the periplasm and media at a very high level by the Tat pathway in a manner almost completely dependent on cytoplasmic disulfide formation, by other two putative Tat SPs: those of MdoD and AmiC. In contrast, the TorA SP exports YebF at a low level.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae. It consists of the AcrB transporter, which is embedded in the inner membrane, the AcrA adapter located in the periplasm, and the channel protein TolC responsible for the transport of substrates towards the extracellular environment. Besides conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here we report that the AcrAB pump heavily affects the infection process of the LF82 strain, the prototype of Adherent-Invasive Escherichia coli (AIEC) which are highly abundant in the ileal mucosa of Chron disease patients. We found that the deletion of genes encoding AcrA and/or AcrB leads to decreased survival of LF82 within macrophages. Ectopic AcrAB expression in a acrAB defective mutant restores the wild type condition. Furthermore, we demonstrate that inhibition of AcrB and replacement of the transporter with an unfunctional AcrB also interfere with bacterial viability inside macrophages. Overall, these data suggest a pivotal role of the AcrAB efflux pump in bacteria-host cell interactions also in AIEC. [ABSTRACT FROM AUTHOR]

The outer membrane (OM) is the defining feature of gram-negative bacteria and is an essential organelle. Accordingly, OM assembly pathways and their essential protein components are conserved throughout all gram-negative species. Lipoprotein trafficking lies at the heart of OM assembly since it supplies several different biogenesis machines with essential lipoproteins. The Escherichia coli Lol trafficking pathway relies on an inner membrane LolCDE transporter that transfers newly made lipoproteins to the chaperone LolA, which rapidly traffics lipoproteins across the periplasm to LolB for insertion into the OM. Strikingly, many gram-negative species (like Caulobacter vibrioides) do not produce LolB, yet essential lipoproteins are still trafficked to the OM. How the final step of trafficking occurs in these organisms has remained a long-standing mystery. We demonstrate that LolA from C. vibrioides can complement the deletion of both LolA and LolB in E. coli, revealing that this protein possesses both chaperone and insertion activities. Moreover, we define the region of C. vibrioides LolA that is responsible for its bifunctionality. This knowledge enabled us to convert E. coli LolA into a similarly bifunctional protein, capable of chaperone and insertion activities. We propose that a bifunctional LolA eliminates the need for LolB. Our findings provide an explanation for why some gram-negative species have retained an essential LolA yet completely lack a dedicated LolB protein. [ABSTRACT FROM AUTHOR]

The treatment of infections by Gram-negative bacteria remains a difficult clinical challenge. In the light of the dearth of discovery of novel antibiotics, one strategy that is being explored is the use of adjuvants to enhance antibacterial activities of existing antibiotics. One such adjuvant is bulgecin A, which allows for the lowering of minimal-inhibitory concentrations for β-lactam antibiotics. We have shown that bulgecin A inhibits three of the pseudomonal lytic transglycosylases in its mode of action, yet high concentrations are needed for potentiation activity. Herein, we document that bulgecin A is not a substrate for pseudomonal efflux pumps, whose functions could have been a culprit in the need for high concentrations. We present evidence that the penetration barrier into the periplasm is at the root of the need for high concentrations of bulgecin A in its potentiation of β-lactam antibiotics. [ABSTRACT FROM AUTHOR]

[Display omitted] • PFS can serve as a potential electron acceptor in the process of nitrogen removal. • Fe3+ as an extracellular electron acceptor compensated nitrite deficiency in Anammox. • Feammox-Anammox combined system achieved nitrogen removal of 85 % at NO 2 –-N/NH 4 +-N of 0.5. • Proportion of ammonia loss by extracellular electron transfer increased from 0.3% to 45%. • Ca. Brocadia and Ca.Kuenenia alternately dominated intracellular and extracellular electron transfer. In addressing the issue of the unstable supply of electron acceptors (nitrite) during Anammox, researchers have often overlooked that the electron transfer process in anammox bacteria (AnAOB) is not restricted to the intracellular, extracellular electron transfer can also serve as an alternative pathway. This study utilized Fe3+ as an adjunctive electron acceptor, coupling Anammox with ferric iron reduction (Feammox) by introducing polymeric ferric sulfate (PFS) in the Anammox system. This approach achieved effective nitrogen removal even when nitrite was deficient. At a NO 2 –-N to NH 4 +-N ratio of 0.5, the Feammox-Anammox combined system maintained stable nitrogen removal under the dual electron acceptor, with total nitrogen and NH 4 +-N removal efficiency of 85 % and 91 %, respectively. The contribution of Feammox and Anammox to NH 4 +-N removal was 45 % and 55 %, respectively. TEM-EDS confirmed the presence of iron elements across the periphery, periplasm, and cytoplasm of AnAOB, suggesting the involvement of PFS in the metabolic and electron transfer processes. Notably, the introduction of Fe3+ and the limitation of nitrite availability resulted in increased relative abundances of FeOB (Thermomonas) and FeRB (Ignavibacterium) to 30 % and 3.6 %, respectively, indicating that the electron transfer process with Fe3+ as the electron acceptor was continuously intensified, as well as a cycle of Fe3+ / Fe2+ was established. Due to unique nitrogen metabolism mode and high Fe3+ tolerance, the abundance of Candidatus_Brocadia was enriched up to 7.2 %, which alternately dominated the Anammox process with Candidatus_Kuenenia , providing favorable conditions for AnAOB to carry out the extracellular electron transfer process. These findings provide a novel strategy for improving the stability and efficiency of the Anammox process under deficient nitrite supply. [ABSTRACT FROM AUTHOR]

Abstract Background Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli). Results The BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity. Conclusions The present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.

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.

Surface layers (S-layers) are highly ordered coats of proteins localized on the cell surface of many bacterial species. In these structures, one or more proteins form elementary units that self-assemble into a crystalline monolayer tiling the entire cell surface. Here, the cell envelope of the radiation-resistant bacterium Deinococcus radiodurans was studied by cryo-electron microscopy, finding the crystalline regularity of the S-layer extended into the layers below (outer membrane, periplasm, and inner membrane). The cell envelope appears to be highly packed and resulting from a three-dimensional crystalline distribution of protein complexes organized in close continuity yet allowing a certain degree of free space. The presented results suggest how S-layers, at least in some species, are mesoscale assemblies behaving as structural and functional scaffolds essential for the entire cell envelope. [ABSTRACT FROM AUTHOR]

The molecular chaperones HdeA and HdeB of the Escherichia coli (E. coli) periplasm protect client proteins from acid denaturation through a unique mechanism that utilizes their acid denatured states to bind clients. We previously demonstrated that the active, acid-denatured form of HdeA is also prone to forming inactive, amyloid fibril-like aggregates in a pH-dependent, reversible manner. In this study, we report that HdeB also displays a similar tendency to form fibrils at low pH. HdeB fibrils were observed at pH < 3 in the presence of NaCl. Similar to HdeA, HdeB fibrils could be resolubilized by a simple shift to neutral pH. In the case of HdeB, however, we found that after extended incubation at low pH, HdeB fibrils were converted into a form that could not resolubilize at pH 7. Fresh fibrils seeded from these "transformed" fibrils were also incapable of resolubilizing at pH 7, suggesting that the transition from reversible to irreversible fibrils involved a specific conformational change that was transmissible through fibril seeds. Analyses of fibril secondary structure indicated that HdeB fibrils retained significant alpha helical content regardless of the conditions under which fibrils were formed. Fibrils that were formed from HdeB that had been treated to remove its intrinsic disulfide bond also were incapable of resolubilizing at pH 7, suggesting that certain residual structures that are retained in acid-denatured HdeB are important for this protein to recover its soluble state from the fibril form. [ABSTRACT FROM AUTHOR]

Beta-lactam antibiotics are often the treatment of choice for serious bacterial infections. In a previous screen for novel genetic mediators affecting beta-lactam susceptibility, we discovered that deletion of ydgH , a conserved gene of unknown function, leads to increased resistance to beta-lactams, as well as increased susceptibility to detergent compounds. Here, we further characterize YdgH in Serratia marcescens, Enterobacter cloacae , and Escherichia coli using a combination of biochemical and cell biological approaches. We find that YdgH fractionates with periplasmic proteins, and this periplasmic localization is necessary for its function. Using purified recombinant protein, we demonstrate that YdgH is a relatively compact, globular monomer. The YdgH polypeptide contains three tandem DUF1471 domains. In a Δ ydgH background, overexpression of polypeptides containing both the second and the third, but not the first DUF1471 domain, is necessary to rescue the deletion phenotype. To determine how YdgH function influences beta-lactam and detergent susceptibility, we tested several targeted hypotheses. We found that deletion of ydgH neither affects ompC or ompF transcript levels , nor does it alter the processing of lipopolysaccharide, nor does it activate the sigma E regulon alone or in combination with mutations in other periplasmic proteins. Finally, we delineate the results of a genetic screen for spontaneous mutants that complement the detergent susceptibility phenotype, the results of which may fuel the further studies that are necessary to determine the precise role YdgH plays in bacterial physiology.IMPORTANCEBeta-lactams such as penicillins and cephalosporins are the most commonly prescribed antibiotics for serious bacterial infections. Increasing antibiotic resistance threatens their effectiveness. We previously identified the uncharacterized gene ydgH as a modifier of beta-lactam susceptibility in Gram-negative bacteria. To begin to understand the specific role of YdgH, in this study, we perform initial characterizations of this protein. We also test hypotheses as to how the function of YdgH contributes to beta-lactam physiology.

The Tol-Pal proteins stabilize the outer membrane during cell division in many Gram-negative bacteria, including Escherichia coli . Pal is an outer membrane lipoprotein that can bind peptidoglycan. It accumulates at the septum during division by a mobilization-and-capture mechanism. This work further substantiates and extends knowledge of Pal's localization in E. coli using immunolabelling; this method enables the detection of endogenous proteins. The midcell localization of Pal and TolB, as seen with fluorescent protein fusions, during cell division, was confirmed. The retention of Pal in newly formed cell poles seemed to persist longer than observed with fluorescent Pal fusions. The concentration of endogenous Pal during the cell division cycle fluctuated: it decreased initially (to half the fluorescence concentration (32.1 au µm -3 ) of the maximum (64.1 au µm -3 ) reached during the cell cycle) and then increased during the second half of the cell division cycle. We probed for possible regulators and proposed two new putative regulators of Pal. By deleting the periplasmic protease, Prc decreased the total Pal abundance (to ~65% of the fluorescence concentration in WT cells) and affected its concentration fluctuation during the cell cycle. This suggests that Prc controls a cell division stage-specific regulator of Pal. Immunolabelling also supported the prediction that the small RNA MicA suppresses Pal expression (the fluorescence concentration of Pal in cells without MicA is double that of Pal in WT cells). However, the regulation by MicA occurred in a cell cycle-independent manner. All these findings urge further research on the tight regulation of the dividing cell envelope stability., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors.)

Many Gram-negative bacteria use CdiA effector proteins to inhibit the growth of neighboring competitors. CdiA transfers its toxic CdiA-CT region into the periplasm of target cells, where it is released through proteolytic cleavage. The N-terminal cytoplasm-entry domain of the CdiA-CT then mediates translocation across the inner membrane to deliver the C-terminal toxin domain into the cytosol. Here, we show that proteolysis not only liberates the CdiA-CT for delivery, but is also required to activate the entry domain for membrane translocation. Translocation function depends on precise cleavage after a conserved VENN peptide sequence, and the processed ∆VENN entry domain exhibits distinct biophysical and thermodynamic properties. By contrast, imprecisely processed CdiA-CT fragments do not undergo this transition and fail to translocate to the cytoplasm. These findings suggest that CdiA-CT processing induces a critical structural switch that converts the entry domain into a membrane-translocation competent conformation. Contact-dependent growth inhibition (CDI) is an important mechanism of bacterial competition. Here, Bartelli et al. show that proteolytic processing of a CDI toxin induces a conformational switch required for translocation into target bacteria. [ABSTRACT FROM AUTHOR]

The Salmonella flagellar secretion apparatus is a member of the type III secretion (T3S) family of export systems in bacteria. After completion of the flagellar motor structure, the hook-basal body (HBB), the flagellar T3S system undergoes a switch from early to late substrate secretion, which results in the expression and assembly of the external, filament propeller-like structure. In order to characterize early substrate secretion-signals in the flagellar T3S system, the FlgB, and FlgC components of the flagellar rod, which acts as the drive-shaft within the HBB, were subject to deletion mutagenesis to identify regions of these proteins that were important for secretion. The β-lactamase protein lacking its Sec-dependent secretion signal (Bla) was fused to the C-terminus of FlgB and FlgC and used as a reporter to select for and quantify the secretion of FlgB and FlgC into the periplasm. Secretion of Bla into the periplasm confers resistance to ampicillin. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 of FlgB decreased FlgB secretion levels while deleting amino acid 6 through 14 diminished FlgC secretion levels. Further PCR-directed mutagenesis indicated that amino acid F45 of FlgB was critical for secretion. Single amino acid mutagenesis revealed that all amino acid substitutions at F45 of FlgB position impaired rod assembly, which was due to a defect of FlgB secretion. An equivalent F49 position in FlgC was essential for assembly but not for secretion. This study also revealed that a hydrophobic patch in the cleaved C-terminal domain of FlhB is critical for recognition of FlgB at F45. Author summary: Type III secretion (T3S) is the means by which proteins are secreted from the bacterial cytoplasm to build flagella for motility and injectisome structures that facilitate pathogenesis. T3S is the only secretion system known to date that undergoes a secretion-specificity switch. For the assembly of the bacterial flagellum, the T3S system initially secretes early substrates to build the hook-basal body (HBB), which is the main component that makes up the flagellar motor. Upon HBB completion, the flagellar T3S system becomes specific for late substrates, which make up the long external filament that acts as the propeller of the motility organelle. This work identifies important sites of interaction between an early substrate, FlgB and a target site at the cytoplasmic base of T3S apparatus. A second early substrate, FlgC, lacks the targeting interaction found for FlgB suggesting a mechanism that distinguishes early substrates, and may indicate an order to early substrate secretion to facilitate the order of protein subunit assembly for the flagellum. [ABSTRACT FROM AUTHOR]

BackgroundAs regulators of multifunctional metalloproteinases including MMP, ADAM and ADAMTS families, tissue inhibitors of metalloproteinases (TIMPs) play a pivotal role in extracellular matrix remodeling, which is involved in a wide variety of physiological processes. Since abnormal metalloproteinase activities are related to numerous diseases such as arthritis, cancer, atherosclerosis, and neurological disorders, TIMPs and their engineered mutants hold therapeutic potential and thus have been extensively studied. Traditional productions of functional TIMPs and their N-terminal inhibitory domains (N-TIMPs) rely on costly and time-consuming insect and mammalian cell systems, or tedious and inefficient refolding from denatured inclusion bodies. The later process is also associated with heterogeneous products and batch-to-batch variation.ResultsIn this study, we developed a simple approach to directly produce high yields of active TIMPs in the periplasmic space of Escherichia coli without refolding. Facilitated by disulfide isomerase (DsbC) co-expression in protease-deficient strain BL21 (DE3), N-TIMP-1/-2 and TIMP-2 which contain multiple disulfide bonds were produced without unwanted truncations. 0.2-1.4 mg purified monomeric TIMPs were typically yielded per liter of culture media. Periplasmically produced TIMPs exhibited expected inhibition potencies towards MMP-1/2/7/14, and were functional in competitive ELISA to elucidate the binding epitopes of MMP specific antibodies. In addition, prepared N-TIMPs were fully active in a cellular context, i.e. regulating cancer cell morphology and migration in 2D and 3D bioassays.ConclusionPeriplasmic expression in E. coli is an excellent strategy to recombinantly produce active TIMPs and N-TIMPs.

Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the cus operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

Microbially mediated Fe(II) oxidation has a great potential for attenuating arsenic (As) mobility in an anoxic groundwaters. Green rust (GR), a common Fe(II)-bearing phase in such environments, could be easily oxidized into Fe (oxyhydr)oxides through microbial activity. This study focused on Acidovorax sp. strain BoFeN1, an anaerobic nitrate-reducing Fe(II)-oxidizing (NRFO) bacterium, to promote the transformation of GR. In biotic GR transformation experiments, magnetite formation occurred at [As] ini = 5 mg/L while lepidocrocite and goethite were formed at [As] ini = 10 mg/L. In the absence of bacterium, the GR persisted throughout the 120-h experiment. Meanwhile, with the addition of strain BoFeN1, the final aqueous As concentration significantly decreased from 0.237 to 0.004 mg/L (C 0 = 5 mg/L) and from 1.457 to 0.096 mg/L (C 0 = 10 mg/L) at 120 h. It was indicated that strain BoFeN1 played a crucial role in promoting the GR transformation and enhancing As immobilization. Further investigations revealed that the role of strain BoFeN1 extended beyond Fe-oxidation. With nitrite (the intermediate of nitrate bioreduction) as oxidizer, lepidocrocite/goethite were formed in the chemical-oxidation system, excluding magnetite. In the Bio - [As] ini = 5 mg/L, the occurrence of lepidocrocite via the bio-oxidation of Fe(II) in GR at 24 h, along with the metabolism of strain BoFeN1 reducing nitrate accompanied with H+ consumption, it should be reasonably deduced that the alkaline micro-environment of periplasm induced by strain BoFeN1 were vital for the transformation of lepidocrocite to magnetite triggered by trace Fe(II). However, in the Bio - [As] ini = 10 mg/L, more As adsorbed on GR inhibiting the adsorption of bacterium, so the alkaline micro-environment had no obvious effect on such transformation. This study helps to understand the interdependence between GR and anaerobic NRFO bacterium, and provides a new perspective for more effective As remediation strategies in anoxic groundwaters. [Display omitted] • Acidovorax sp. strain BoFeN1 promoted the transformation of GR in presence of As and increased As immobilization. • Bacterium adsorbed on GR to oxidize Fe(II) in GR forming lepidocrocite during the transformation of GR to magnetite. • The alkaline micro-environment induced by strain BoFeN1 was vital for the transformation of GR to magnetite. [ABSTRACT FROM AUTHOR]

Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Providencia stuartii is a highly social pathogen responsible for nosocomial chronic urinary tract infections. The bacterium indeed forms floating communities of cells (FCC) besides and prior-to canonical surface-attached biofilms (SAB). Within P. stuartii FCC, cells are riveted one to another owing to by self-interactions between its porins, viz. Omp-Pst1 and Omp-Pst2. In pathophysiological conditions, P. stuartii is principally exposed to high concentrations of urea, ammonia, bicarbonate, creatinine and to large variations of pH, questioning how these environmental cues affect socialization, and whether formation of SAB and FCC protects cells against those. Results from our investigations indicate that FCC and SAB can both form in the urinary tract, endowing cells with increased resistance and fitness. They additionally show that while Omp-Pst1 is the main gateway allowing penetration of urea, bicarbonate and ammonia into the periplasm, expression of Omp-Pst2 enables resistance to them. [ABSTRACT FROM AUTHOR]

Auto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of E. coli compared to inducer-dependent systems. In this study, we investigated the phosphate (PO4)-sensitive phoA expression system (pAT) for the production of a recombinant model antigen-binding fragment (Fab) in the periplasm of E. coli in detail. We explored the impact of non-limiting and limiting PO4 conditions on strain physiology as well as Fab productivity. We compared different methods for extracellular PO4 detection, identifying automated colorimetric measurement to be most suitable for at-line PO4 monitoring. We showed that PO4 limitation boosts phoA-based gene expression, however, the product was already formed at non-limiting PO4 conditions, indicating leaky expression. Furthermore, cultivation under PO4 limitation caused physiological changes ultimately resulting in a metabolic breakdown at PO4 starvation. Finally, we give recommendations for process optimization with the phoA expression system. In summary, our study provides very detailed information on the E. coli phoA expression system, thus extending the existing knowledge of this system, and underlines its high potential for the successful production of periplasmic products in E. coli. [ABSTRACT FROM AUTHOR]

On the periplasmic side of LacY, two conserved Gly-Gly pairs in helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) allow close packing of each helix pair in the outward (periplasmic)-closed conformation. Previous studies demonstrate that replacing one Gly residue in each Gly-Gly pair with Trp leads to opening of the periplasmic cavity with abrogation of transport activity, but an increased rate of galactoside binding. To further investigate the role of the Gly-Gly pairs, 11 double-replacement mutants were constructed for each pair at positions 46 (helix II) and 262 (helix VIII). Replacement with Ala or Ser results in decreased but significant transport activity, while replacements with Thr, Val, Leu, Asn, Gln, Tyr, Trp, Glu, or Lys exhibit very little or no transport. Remarkably, however, the double mutants bind galactoside with affinities 10-20-fold higher than that of the pseudo-WT or WT LacY. Moreover, site-directed alkylation of a periplasmic Cys replacement indicates that the periplasmic cavity becomes readily accessible in the double-replacement mutants. Molecular dynamics simulations with the WT and double-Leu mutant in the inward-open/outward-closed conformation provide support for this interpretation.

Background: Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli). Results: The BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity. Conclusions: The present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research. [ABSTRACT FROM AUTHOR]

Understanding the evolution of metallo-β-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and opportunistic bacteria. Presently, these MBL-producing pathogens are linked to high rates of morbidity and mortality worldwide. However, the study of the biochemical and biophysical features of MBLs in vitro provides an incomplete picture of their evolutionary potential, since this limited and artificial environment disregards the physiological context where evolution and selection take place. Herein, we describe recent efforts aimed to address the evolutionary traits acquired by different clinical variants of MBLs in conditions mimicking their native environment (the bacterial periplasm) and considering whether they are soluble or membrane-bound proteins. This includes addressing the metal content of MBLs within the cell under zinc starvation conditions and the context provided by different bacterial hosts that result in particular resistance phenotypes. Our analysis highlights recent progress bridging the gap between in vitro and in-cell studies. [ABSTRACT FROM AUTHOR]

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise nonessential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation [ABSTRACT FROM AUTHOR]

The symport of lactose and H+ is an important physiological process in E. coli, for it is closely related to cellular energy supply. In this paper, we review, extend and analyse a newly proposed cotransport model that takes the "leakage" phenomenon (uncoupled particle translocation) into account and also satisfies the static head equilibrium condition. Then, we use the model to study the equilibrium properties, including equilibrium solution and the time required to reach equilibrium, of the symport process of E. coli LacY protein, when varying the parameters of the initial state of cotransport system. It can be found that in our extended model, H+ and lactose will reach their equilibrium state separately, and when "leakage" exists, it linearly affects the equilibrium solution, which is a useful property that the original model does not have. We later investigated the effect of the volume of periplasm and cytoplasm on the equilibrium properties. For a certain E. coli cell, as it continues to lose water and contract, the time for cytoplasm pH to be stabilized by symport increases monotonically when the cell survives. Finally, we reproduce the experimental data from a literature to verify the validity of the extension in this symport process. The above phenomena and other findings in this paper may help us to not only further validate or improve the model, but also deepen our understanding of the cotransport process of E. coli LacY protein. [ABSTRACT FROM AUTHOR]

Objective To investigate the function of general chaperones in the secretion of two-partner-secretion (TPS) protein across the inner membrane to the periplasm of Gram-negative bacteria. Methods The general chaperones were knocked-out and the strains were used to prepare spheroplast to see the effects on the secretion of FhaB*, which is a substrate of the representative model TPS substrates FhaB/FhaC system. Results Single knock-out of chaperones Skp,DegP,PpiD,YfgM and double knock-out of PpiD/YfgM has no or little effect on the secretion of FhaB* into the periplasm, while single knock-out of SurA or double knock-out of Skp/DegP severely affect the secretion of FhaB*. Conclusions There may be two molecular chaperone pathways mediated by SurA or by both SKP and DegP in Gram-negative bacteria, which can mediate the substrate protein of the two-partner-secretion system secretes into the peripheral space.

Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C‐terminal his‐tag added for subsequent purification. Our research group has proposed the small metal‐binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB‐SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2‐11 cells, was equivalent to commercial growth hormone at 50 ng·mL−1. Therefore, we strongly recommend the use of PelB‐SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli.