Control of biofilm formation by an Agrobacterium tumefaciens pterin-binding periplasmic protein conserved among diverse Proteobacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Computational design of Periplasmic binding protein biosensors guided by molecular dynamics.

Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein's output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.

Discovery of periplasmic solute binding proteins with specificity for ketone bodies: ß-hydroxybutyrate binding proteins.

Polyhydroxyalkanoates are a class of biodegradable, thermoplastic polymers which represent a major carbon source for various bacteria. Proteins which mediate the translocation of polyhydroxyalkanoate breakdown products, such as ß-hydroxybutyrate (BHB)-a ketone body which in humans serves as an important biomarker, have not been well characterized. In our investigation to screen a solute-binding protein (SBP) which can act as a suitable recognition element for BHB, we uncovered insights at the intersection of bacterial metabolism and diagnostics. Herein, we identify SBPs associated with putative ATP-binding cassette transporters that specifically recognize BHB, with the potential to serve as recognition elements for continuous quantification of this analyte. Through bioinformatic analysis, we identified candidate SBPs from known metabolizers of polyhydroxybutyrate-including proteins from Cupriavidus necator, Ensifer meliloti, Paucimonas lemoignei, and Thermus thermophilus. After recombinant expression in Escherichia coli, we demonstrated with intrinsic tryptophan fluorescence spectroscopy that four candidate proteins interacted with BHB, ranging from nanomolar to micromolar affinity. Tt.2, an intrinsically thermostable protein from Thermus thermophilus, was observed to have the tightest binding and specificity for BHB, which was confirmed by isothermal calorimetry. Structural analyses facilitated by AlphaFold2, along with molecular docking and dynamics simulations, were used to hypothesize key residues in the binding pocket and to model the conformational dynamics of substrate unbinding. Overall, this study provides strong evidence identifying the cognate ligands of SBPs which we hypothesize to be involved in prokaryotic cellular translocation of polyhydroxyalkanoate breakdown products, while highlighting these proteins' promising biotechnological application.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

An automatic glucose monitoring system based on periplasmic binding proteins for online bioprocess monitoring.

Glucose is one of the most vital nutrients in all living organisms, so its monitoring is critical in healthcare and bioprocessing. Enzymatic sensors are more popular as a technology solution to meet the requirement. However, periplasmic binding proteins have been investigated extensively for their high sensitivity, enabling microdialysis sampling to replace existing complex and expensive glucose monitoring solutions based on enzymatic sensors. The binding proteins are used as optical biosensors by introducing an environment-sensitive fluorophore to the protein. The biosensor's construction, characterization, and potential application are well studied, but a complete glucose monitoring system based on it is yet to be reported. This work documents the development of the first glucose sensor prototype based on glucose binding protein (GBP) for automatic and continuous glucose measurements. The development includes immobilizing the protein into reusable chips and a low-cost solution for non-invasive glucose sampling in bioprocesses using microdialysis sampling technique. A program was written in LabVIEW to accompany the prototype for the complete automation of measurement. The sampling technique allowed glucose measurements of a few micromolar to 260 mM glucose levels. A thorough analysis of the sampling mode and the device's performance was conducted. The reported measurement accuracy was 81.78%, with an RSD of 1.83%. The prototype was also used in online glucose monitoring of E. coli cell culture. The mode of glucose sensing can be expanded to the measurement of other analytes by switching the binding proteins.

Molecular handcraft of a well-folded protein chimera.

Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous 'copy-paste' processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/ß-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7 µM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter.

The outer membrane (OM) is an essential organelle of Gram-negative bacteria. Lipoproteins are key to building the OM, performing essential functions in several OM assembly machines. Lipoproteins mature in the inner membrane (IM) and are then trafficked to the OM. In Escherichia coli, the LolCDE transporter is needed to extract lipoproteins from the IM to begin trafficking. Lipoproteins are then transferred from LolCDE to the periplasmic chaperone LolA which ferries them to the OM for insertion by LolB. LolA recruitment by LolC is an essential trafficking step. Structural and biochemical studies suggested that two regions (termed Hook and Pad) within a periplasmic loop of LolC worked in tandem to recruit LolA, leading to a bipartite model for recruitment. Here, we genetically examine the LolC periplasmic loop in vivo using E. coli. Our findings challenge the bipartite interaction model. We show that while the Hook is essential for lipoprotein trafficking in vivo, lipoproteins are still efficiently trafficked when the Pad residues are inactivated. We show with AlphaFold2 multimer modeling that Hook:LolA interactions are likely universal among diverse Gram-negative bacteria. Conversely, Pad:LolA interactions vary across phyla. Our in vivo data redefine LolC:LolA recruitment into a hierarchical interaction model. We propose that the Hook is the major player in LolA recruitment, while the Pad plays an ancillary role that is important for efficiency but is ultimately dispensable. Our findings expand the understanding of a fundamental step in essential lipoprotein trafficking and have implications for efforts to develop new antibacterials that target LolCDE.IMPORTANCEResistance to current antibiotics is increasingly common. New antibiotics that target essential processes are needed to expand clinical options. For Gram-negative bacteria, their cell surface-the outer membrane (OM)-is an essential organelle and antibiotic barrier that is an attractive target for new antibacterials. Lipoproteins are key to building the OM. The LolCDE transporter is needed to supply the OM with lipoproteins and has been a focus of recent antibiotic discovery. In vitro evidence recently proposed a two-part interaction of LolC with LolA lipoprotein chaperone (which traffics lipoproteins to the OM) via "Hook" and "Pad" regions. We show that this model does not reflect lipoprotein trafficking in vivo. Only the Hook is essential for lipoprotein trafficking and is remarkably robust to mutational changes. The Pad is non-essential for lipoprotein trafficking but plays an ancillary role, contributing to trafficking efficiency. These insights inform ongoing efforts to drug LolCDE.

Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking.

The envelope of Escherichia coli contains approximately 100 different species of lipoproteins, most of which are localized to the inner leaflet of the outer membrane. The localization of lipoprotein (Lol) system, consisting of five Lol proteins, is responsible for the trafficking of lipoproteins to the outer membrane. LolCDE binds to lipoproteins destined for the outer membrane and transfers them to the periplasmic chaperone LolA. Although the cryo-EM structures of E. coli LolCDE have been reported, the mechanisms by which outer membrane lipoproteins are transferred to LolA remain elusive. In this study, we investigated the interaction between LolCDE and lipoproteins using site-specific photo-crosslinking. We introduced a photo-crosslinkable amino acid into different locations across the four helices which form the central lipoprotein-binding cavity, and identified domains that crosslink with peptidoglycan-associated lipoprotein (Pal) in vivo. Using one of the derivatives containing the photo-crosslinkable amino acid, we developed an in vitro system to analyze the binding of lipoproteins to LolCDE. Our results indicate that compound 2, a LolCDE inhibitor, does not inhibit the binding of lipoproteins to LolCDE, but rather promotes the dissociation of bound lipoproteins from LolCDE.

Metabolic network and proteomic expression perturbed by cyclosporine A to model microbe Escherichia coli.

Cyclosporine A (CsA) is a model drug that has caused great concern due to its widespread use and abuse in the environment. However, the potential harm of CsA to organisms also remains largely unknown, and this issue is exceptionally important for the health risk assessment of antibiotics. To address this concern, the crosstalk between CsA stress and cellular metabolism at the proteomic level in Escherichia coli was investigated and dissected in this study. The results showed that CsA inhibited E. coli growth in a time-dependent manner. CsA induced reactive oxygen species (ROS) overproduction in a dose- and time-dependent manner, leading to membrane depolarization followed by cell apoptosis. In addition, translation, the citric acid cycle, amino acid biosynthesis, glycolysis and responses to oxidative stress and heat were the central metabolic pathways induced by CsA stress. The upregulated proteins, including PotD, PotF and PotG, controlled cell growth. The downregulated proteins, including SspA, SspB, CstA and DpS, were regulators of self-feedback during the starvation process. And the up- and downregulated proteins, including AtpD, Adk, GroS, GroL and DnaK, controlled energy production. These results provide an important reference for the environmental health risk assessment of CsA.

Identification of interaction partners and function analysis of new splicing product of human LMO2 gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the interaction partners of a new splicing product of LMO2 gene (LMO2-C), and study its function in K562 cells.</p><p><b>METHODS</b>Maltose binding protein (MBP) pull down and mammalian two-hybrid assay (MTHA) were used to identify the interaction partners of LMO2-C in K562 cells. Semiquantitative RT-PCR was used to detect the expression of hematopoietic specific gene glycoprotein (GPA) in K562 cells.</p><p><b>RESULTS</b>MBP-LMO2-C fusion protein was expressed and purified in soluble form successfully. Endogenous GATA1 and LDB1 proteins were confirmed to bind to LMO2-C by MBP pull down analysis. The MTHA also showed that LMO2-C had comparable binding affinities to LDB1 with LMO2-L, and over expression of LMO2-C prevented LMO2-L from binding to LDB1, the inhibition rate being (81.13 +/- 0.68)%. RT-PCR results showed that the expression level of GPA was reduced [(51.00 +/- 1.58)%] in K562 cells while LMO2-C overexpressed.</p><p><b>CONCLUSION</b>LMO2-C can bind endogenous GATA1 and LDB1 protein in K562 cells and down regulates the expression of GPA.</p>

Sequence analysis on sorbitol fermentation related genes in Vibrio cholerae / 中华流行病学杂志

<p><b>OBJECTIVE</b>To Investigate the differences of sorbitol fermentation related genes and optimize molecular analysis method for distinguishing an epidemic with nonepidemic strains of Vibrio cholerae.</p><p><b>METHODS</b>Sequence analysis on four genes of sugar fermentation stimulation protein, periplasmic maltose-binding protein, periplasmic phosphate-binding protein and periplasmic amino acid-binding protein.</p><p><b>RESULTS</b>In this study, the following data was noticed: for O1 serogroup El Tor biotype V. cholerae, twenty-four epidemic and eight nonepidemic strains were chosen; For O139 serogroup V. cholerae, five epidemic and four nonepidemic strains were chosen. With those genes of sugar fermentation stimulation protein, there were three point mutations. The 106th, 150th, 378th oligonucleotide in epidemic strains were A, A and T, comparing to the nonepidemic strains which were G, G and C. When comparing the protein sequences, epidemic strains had a Threonine at 36th amino acid, whereas nonepidemic strains had an Alanine. The results in O139 serogroup were consistent with those in O1 serogroup El Tor biotype strains. Another two point mutations were found in the genes of periplasmic maltose-binding protein. The 999th, 1003rd oligonucleotides in epidemic strains were A and C, while in nonepidemic which were G and T. For the gene of periplasmic amino acid-binding protein, two point mutations were noticed. The 504th and 690th oligonucleotides in epidemic strains were T and C, but were C and T in nonepidemic. However, no amino acid differences were found in periplasmic maltose-binding protein and periplasmic amino acid-binding protein. For periplasmic amino acid-binding protein gene, there was no difference on oligonucleotide between epidemic and nonepidemic strains.</p><p><b>CONCLUSION</b>Results suggested that SNPs in these genes might serve as a useful tool to distinguish the epidemic strains from nonepidemic strains. The 36th amino acid mutation of sugar fermentation stimulation protein in epidemic and nonepidemic strains might change the activity of the protein which might be associated with sorbitol fermentation.</p>