Capture-and-release of a sulfoquinovose-binding protein on sulfoquinovose-modified agarose.

The solute-binding protein (SBP) components of periplasmic binding protein-dependent ATP-binding cassette (ABC)-type transporters often possess exquisite selectivity for their cognate ligands. Maltose binding protein (MBP), the best studied of these SBPs, has been extensively used as a fusion partner to enable the affinity purification of recombinant proteins. However, other SBPs and SBP-ligand based affinity systems remain underexplored. The sulfoquinovose-binding protein SmoF, is a substrate-binding protein component of the ABC transporter cassette in Agrobacterium tumefaciens involved in importing sulfoquinovose (SQ) and its derivatives for SQ catabolism. Here, we show that SmoF binds with high affinity to the octyl glycoside of SQ (octyl-SQ), demonstrating remarkable tolerance to extension of the anomeric substituent. The 3D X-ray structure of the SmoF·octyl-SQ complex reveals accommodation of the octyl chain, which projects to the protein surface, providing impetus for the synthesis of a linker-equipped SQ-amine using a thiol-ene reaction as a key step, and its conjugation to cyanogen bromide modified agarose. We demonstrate the successful capture and release of SmoF from SQ-agarose resin using SQ as competitive eluant, and selectivity for release versus other organosulfonates. We show that SmoF can be captured and purified from a cell lysate, demonstrating the utility of SQ-agarose in capturing SQ binding proteins from complex mixtures. The present work provides a pathway for development of 'capture-and-release' affinity resins for the discovery and study of SBPs.

Cryo-EM structure of the Agrobacterium tumefaciens T4SS-associated T-pilus reveals stoichiometric protein-phospholipid assembly.

Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer.

Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen.

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.

An NAD-Specific 6-Hydroxy-3-Succinoyl-Semialdehyde-Pyridine Dehydrogenase from Nicotine-Degrading Agrobacterium tumefaciens Strain S33.

Agrobacterium tumefaciens strain S33 can catabolize nicotine via a hybrid of the pyridine and pyrrolidine pathways. Most of the enzymes involved in this biochemical pathway have been identified and characterized, except for the one catalyzing the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. Based on a previous genomic and transcriptomic analysis, an open reading frame (ORF) annotated to encode aldehyde dehydrogenase (Ald) in the nicotine-degrading cluster was predicted to be responsible for this step. In this study, we heterologously expressed the enzyme and identified its function by biochemical assay and mass spectrum analysis. It was found that Ald catalyzes the NAD-specific dehydrogenation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. With the nonhydroxylated analog 3-succinoyl-semialdehyde-pyridine (SAP) as a substrate, Ald had a specific activity of 10.05 U/mg at pH 9.0 and apparent Km values of around 58.68 µM and 0.41 mM for SAP and NAD+, respectively. Induction at low temperature and purification and storage in low-salt buffers were helpful to prevent its aggregation and precipitation. Disruption of the ald gene caused a lower growth rate and biomass of strain S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine. Ald has a broad range of substrates, including benzaldehyde, furfural, and acetaldehyde. Recombinant Escherichia coli cells harboring the ald gene can efficiently convert furfural to 2-furoic acid at a specific rate of 0.032 mmol min-1 g dry cells-1, extending the application of Ald in the catalysis of bio-based furan compounds. These findings provide new insights into the biochemical mechanism of the nicotine-degrading hybrid pathway and the possible application of Ald in industrial biocatalysis. IMPORTANCE Nicotine is one of the major toxic N-heterocyclic aromatic alkaloids produced in tobacco plants. Manufacturing tobacco and smoking may lead to some environmental and public health problems. Microorganisms can degrade nicotine by various biochemical pathways, but the biochemical mechanism for nicotine degradation has not been fully elucidated. In this study, we identified an aldehyde dehydrogenase responsible for the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine; this was the only uncharacterized enzyme in the hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33. Similar to the known aldehyde dehydrogenase, the NAD-specific homodimeric enzyme presents a broad substrate range with high activity in alkaline and low-salt-containing buffers. It can catalyze not only the aldehyde from nicotine degradation but also those of benzaldehyde, furfural, and acetaldehyde. It was found that recombinant Escherichia coli cells harboring the ald gene could efficiently convert furfural to valuable 2-furoic acid, demonstrating its potential application for enzymatic catalysis.

Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily.

Aconitase superfamily members catalyze the homologous isomerization of specific substrates by sequential dehydration and hydration and contain a [4Fe-4S] cluster. However, monomeric and heterodimeric types of function unknown aconitase X (AcnX) have recently been characterized as a cis-3-hydroxy-L-proline dehydratase (AcnXType-I) and mevalonate 5-phosphate dehydratase (AcnXType-II), respectively. We herein elucidated the crystal structures of AcnXType-I from Agrobacterium tumefaciens (AtAcnX) and AcnXType-II from Thermococcus kodakarensis (TkAcnX) without a ligand and in complex with substrates. AtAcnX and TkAcnX contained the [2Fe-2S] and [3Fe-4S] clusters, respectively, conforming to UV and EPR spectroscopy analyses. The binding sites of the [Fe-S] cluster and substrate were clearlydifferent from those that were completely conserved in other aconitase enzymes; however, theoverall structural frameworks and locations of active sites were partially similar to each other.These results provide novel insights into the evolutionary scenario of the aconitase superfamilybased on the recruitment hypothesis.

Agrobacterium tumefaciens Growth Pole Ring Protein: C Terminus and Internal Apolipoprotein Homologous Domains Are Essential for Function and Subcellular Localization.

The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth.IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode-polar growth-is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

Type IV secretion systems: Advances in structure, function, and activation.

Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved "core" subunits that respectively elaborate "minimized" systems in Gram-positive or -negative bacteria. However, many "expanded" T4SSs are built from "core" subunits plus numerous others that are system-specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure-function relationships for model Gram-negative bacterial T4SSs, including "minimized" systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and "expanded" systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid-encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long-standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.

An attempt to establish an Agrobacterium-mediated transient expression system in medicinal plants.

Genetic transformation has always been an important method for studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it was time-consuming to obtain a stable transformed hairy root or transgenic plants, which was difficult to satisfy the great demand of researches on medical plant secondary metabolism-related genes. Moreover, Agrobacterium tumefaciens-mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost, and short period. However, presently, researches on medical plant functional genes commonly used stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation. Thus, in this study, we selected the seedlings of Nicotiana benthamiana, Salvia miltiorrhiza, and Prunella vulgaris as the experimental material, with the methods of Agrobacterium tumefaciens injection, fast Agrobacterium-mediated seedling transformation (FAST), and FAST and mechanical damage. The results demonstrated that the injection transient transformation system of pCAMBIA1301 vector mediated by A. tumefaciens and the transient transformation of seedling system were not established in S. miltiorrhiza. In addition, the instantaneous transformation system of N. benthamiana and P. vulgaris seedlings was basically set up by FAST method. Besides, using the method of FAST and mechanical damage, the transient genetic transformation system of P. vulgaris seedlings was established for the first time. A. tumefaciens-mediated transient transformation of seedlings with pEAQ vectors provided an effective way and reference for the further study of functional genes of the medicinal plants N. benthamiana and P. vulgaris.

Identification of a Carboxy-Terminal Glutamine-Rich Domain in Agrobacterium tumefaciens Coupling Protein VirD4 Required for Recognition of T-Strand DNA and Not VirE2 as a Substrate for Transfer to Plant Cells.

Agrobacterium tumefaciens transfers DNA and proteins to a plant cell inciting crown gall tumor disease on most plants. VirD4 targets the DNA and protein substrates to a type IV secretion (T4S) apparatus for translocation into the plant cell. Several bacteria with VirD4 homologs use T4S for intercellular export of microbial macromolecules to eukaryotic and prokaryotic hosts. How the VirD4 proteins recognize the diverse substrates is not well understood. To identify functional domains of A. tumefaciens pTiA6 VirD4, we introduced random 19-codon and targeted 10-codon insertions throughout the coding region. Analysis of 21 mutants showed that only the carboxy-terminal end of VirD4 is tolerant of an insertion. Sequence comparison of VirD4 proteins of Agrobacterium spp. and their close relative, Rhizobium etli, showed that these proteins contain a highly conserved C-terminal end, but the immediate upstream regions share no discernible sequence similarity. The conserved region sequence is rich in the amino acid glutamine (6/13 Q). Using site-specific and deletion mutagenesis, we demonstrated that the conserved Q-rich region is required for VirD4 function and for the specific recognition of VirD2-linked T-strand DNA as a substrate for translocation to plants. The Q-rich region is not required for the transfer of a second A. tumefaciens substrate, VirE2, to plants or a promiscuous Escherichia coli IncQ plasmid to another A. tumefaciens strain. We identified Q-rich sequences at or near the C terminus of several VirD4 homologs, including the E. coli F plasmid TraD. In F TraD, the Q-rich sequence maps to a region required specifically for the conjugative transfer of the F plasmid.

d-Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis.

The bacterial cell wall is composed of membrane layers and a rigid yet flexible scaffold called peptidoglycan (PG). PG provides mechanical strength to enable bacteria to resist damage from the environment and lysis due to high internal turgor. PG also has a critical role in dictating bacterial cell morphology. The essential nature of PG for bacterial propagation, as well as its value as an antibiotic target, has led to renewed interest in the study of peptidoglycan biosynthesis. However, significant knowledge gaps remain that must be addressed before a clear understanding of peptidoglycan synthesis and dynamics is realized. For example, the enzymes involved in the PG biosynthesis pathway have not been fully characterized. Our understanding of PG biosynthesis has been frequently revamped by the discovery of novel enzymes or newly characterized functions of known enzymes. In addition, we do not clearly know how the respective activities of these enzymes are coordinated with each other and how they control the spatial and temporal dynamics of PG synthesis. The emergence of molecular probes and imaging techniques has significantly advanced the study PG synthesis and modification. Prior efforts utilized the specificity of PG-targeting antibiotics and proteins to develop PG-specific probes, such as fluorescent vancomycin and fluorescent wheat germ agglutinin. However, these probes suffer from limitations due to toxic effects toward bacterial cells and poor membrane permeability. To address these issues, we designed and introduced a family of novel molecular probes, fluorescent d-amino acids (FDAAs), which are covalently incorporated into PG through the activities of endogenous bacterial transpeptidases. Their high biocompatibility and PG specificity have made them powerful tools for labeling peptidoglycan. In addition, their enzyme-mediated incorporation faithfully reflects the activity of PG synthases, providing a direct in situ method for studying PG formation during the bacterial life cycle. In this Account, we describe our efforts directed at the development of FDAAs and their derivatives. These probes have enabled for the first time the ability to visualize PG synthesis in live bacterial cells and in real time. We summarize experimental evidence for FDAA incorporation into PG and the enzyme-mediated incorporation pathway. We demonstrate various applications of FDAAs, including bacterial morphology analyses, PG growth model studies, investigation of PG-enzyme correlation, in vitro PG synthase activity assays, and antibiotic inhibition tests. Finally, we discuss the current limitations of the probes and our ongoing efforts to improve them. We are confident that these probes will prove to be valuable tools that will enable the discovery of new antibiotic targets and expand the available arsenal directed at the public health threat posed by antibiotic resistance.

Physical and functional characterization of succinoglycan exopolysaccharide produced by Rhizobium radiobacter CAS from curd sample.

Identification and rheological behaviour of succinoglycan exopolysaccharide (EPS) from Rhizobium radiobacter CAS isolated from curd was published in earlier reports. In current investigation physicochemical and functional properties of CAS EPS has been studied. SEC-MALLS revealed molecular weight (Mw), number molecular weight (Mn) and polydispersity index (PDI) of CAS EPS as 4.05×106g·mol-1, 3.82×106g·mol-1 and, 1.06 respectively. Thermogravimetric analysis showed the high thermal stability of CAS polymer where approximately 77% of CAS hydrocolloid maintain physical integrity and stability with temperature degradation (Td) at 290°C. Scanning electron microscopy and particle size analysis confirmed the porous and hygroscopic nature and 2.049µm size of CAS EPS respectively. Equally important functional properties such as water activity (0.55), water solubility (95%), water contact angle (54.83°), water binding capacity (101g/g), water holding capacity (68.19g/g), oil binding ability (soybean and groundnut oils were found 4.35g/g and 3.68g/g) and swelling index (17.5mL/g) were examined for CAS EPS. Prevention of syneresis has been studied at 1% CAS EPS concentration which significantly prohibited the degree of syneresis of starch. These physico-functional properties make CAS EPS a prominent candidate for food processing and product development sector.

Synthesis of an Undecasaccharide Featuring an Oligomannosidic Heptasaccharide and a Bacterial Kdo-lipid A Backbone for Eliciting Neutralizing Antibodies to Mammalian Oligomannose on the HIV-1 Envelope Spike.

Lipooligosaccharides (LOS) from the bacterium Rhizobium radiobacter Rv3 are structurally related to antigenic mammalian oligomannoses on the HIV-1 envelope glycoprotein spike that are targets for broadly neutralizing antibodies. Here, we prepared a hybrid structure of viral and bacterial epitopes as part of a vaccine design strategy to elicit oligomannose-specific HIV-neutralizing antibodies using glycoconjugates based on the Rv3 LOS structure. Starting from a Kdo2GlcNAc2 tetrasaccharide precursor, a central orthogonally protected mannose trichloroacetimidate donor was coupled to OH-5 of the innermost Kdo residue. To assemble larger glycans, the N-acetylamino groups of the glucosamine units were converted to imides to prevent formation of unwanted imidate byproducts. Blockwise coupling of the pentasaccharide acceptor with an α-(1→2)-linked mannotriosyl trichloroacetimidate donor introduced the D1-arm fragment. Glycosylation of O-6 of the central branching mannose with an α-(1→2)-α-(1→6)-linked mannotriosyl trichloroacetimidate donor unit then furnished the undecasaccharide harboring a D3-arm extension. Global deprotection yielded the 3-aminopropyl ligand, which was activated as an isothiocyanate or adipic acid succinimidoyl ester and conjugated to CRM197. However, representative oligomannose-specific HIV-neutralizing antibodies bound the undecasaccharide conjugates poorly. Possible reasons for this outcome are discussed herein along with paths for improvement.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant's genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed in roots.

Crystal structure of the type VI immunity protein Tdi1 (Atu4351) from Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is a novel multiprotein needle-like apparatus that is distributed widely in Gram-negative bacteria. Bacteria harboring T6SSs inject various effectors into both eukaryotic and prokaryotic cells for interspecies competition or virulence-related processes. The toxicities of the effectors can be neutralized by their cognate immunity proteins. Tde1 (Atu4350)-Tdi1 (Atu4351) has recently been characterized as a T6SS effector-immunity pair in the soil bacterium Agrobacterium tumefaciens and the neutralization mechanism remains unknown. Here, the crystal structure of the immunity protein Tdi1 was determined at 2.40 Šresolution by the single-wavelength anomalous dispersion method. Structural analysis suggested that it is composed of a GAD-like domain and an inserted DUF1851 domain, and both domains show low structural similarities to known structures. There is a positive groove mainly located in the GAD-like domain that may be associated with nucleotide binding. The structure provides a basis for further study of the positive groove as a potential active site.

Affinity sensor for haemoglobin A1c based on single-walled carbon nanotube field-effect transistor and fructosyl amino acid binding protein.

Haemoglobin A1c (HbA1c) is a significant glycaemic marker for diabetes mellitus. The level of HbA1c reflects the mean blood glucose level over the prior 2-3 months and it is useful for the assessment of therapeutic effectiveness and for diagnosis. In this study, we report the label-free affinity sensor for HbA1c based on the chemiresistor-type field-effect transistor, which has a simple sensor configuration. Single-walled carbon nanotubes (SWNTs) were used as the transducing element. The fructosyl amino acid binding protein from Rhizobium radiobacter (SocA), which binds to α-fructosyl amino acid specifically, was used as the biorecognition element for fructosyl valine (FV), the product of the proteolytic hydrolysis of HbA1c. The developed sensor shows the ability to measure as low as 1.2 nM FV, which is 14-fold more sensitive compared to the previously reported fluorescence-based sensor using SocA. This sensor also exhibits high specificity where no significant response is observed from either fructosyl lysine (FK) or glucose, which are potential interferents. FK is the ε-fructosyl amino acid from glycated albumin, another glycated protein, whereas glucose is naturally present at very high concentration in the blood. We propose that the modulation of the surface charges on the SWNTs caused by the conformational change in SocA upon ligand binding leads to the proportionate changes in the number of carriers in the SWNT channel.

Structural basis for two efficient modes of agropinic acid opine import into the bacterial pathogen Agrobacterium tumefaciens.

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic-binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches and affinity measurements, we identified AgtB and its transporter as responsible for agropine uptake in agropine-assimilating agrobacteria. Nonetheless, we showed that AgtB binds agropinic acid with a higher affinity than agropine, and we structurally characterized the agropinic acid-binding mode through three crystal structures at 1.4, 1.74 and 1.9 Šresolution. In the crystallization time course, obtaining a crystal structure of AgtB with agropine was unsuccessful due to the spontaneous lactamization of agropine into agropinic acid. AgaA binds agropinic acid only with a similar affinity in nanomolar range as AgtB. The structure of AgaA bound to agropinic acid at 1.65 Šresolution defines a different agropinic acid-binding signature. Our work highlights the structural and functional characteristics of two efficient agropinic acid assimilation pathways, of which one is also involved in agropine assimilation.

Crystal structure of the Agrobacterium tumefaciens type VI effector-immunity complex.

The type VI secretion system (T6SS) comprises needle-shaped multisubunit complexes that play a role in the microbial defense systems of Gram-negative bacteria. Some Gram-negative bacteria harboring a T6SS deliver toxic effector proteins into the cytoplasm or periplasm of competing bacteria in order to lyse and kill them. To avoid self-cell disruption, these bacteria have cognate immunity proteins that inhibit their toxic effector proteins. T6SS amidase effector protein 4 (Tae4) and T6SS amidase immunity protein 4 (Tai4) are a representative of the toxic effector-immunity pairs of the T6SS. Here, the three-dimensional structures of Tai4 and the Tae4-Tai4 complex from Agrobacterium tumefaciens are reported at 1.55 and 1.9 Šresolution, respectively. A structural comparison with other Tae4-Tai4 homologs revealed similarities and differences in the catalytic and inhibitory mechanisms among the Tae4 and Tai4 family proteins.

RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants.

A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector.

Highly Sensitive and Selective Biosensor for a Disaccharide Based on an AraC-Like Transcriptional Regulator Transduced with Bioluminescence Resonance Energy Transfer.

Sensitive and selective quantification of individual sugars in complex media is technically challenging and usually requires HPLC separation. Accurate measurement without the need for separation would be highly desirable. The measurement of trace levels of lactose in lactose-reduced milk exemplifies the problem, with the added challenge that trace lactose must be measured in the presence of ≈140 mM glucose and galactose, the products of lactase digestion of lactose. Biosensing is an alternative to HPLC, but current biosensing methods, based on coupled-enzyme assays, tend to have poor sensitivity and complex biochemistry and can be time-consuming. We explored a fundamentally different approach, based on identifying a lactose-specific binding protein compatible with photonic transduction. We identified the BgaR transcriptional regulator of Clostridium perfringens, which is highly selective for lactose, as a suitable ligand binding domain and combined it with a bioluminescence energy resonance transfer transduction system. This BRET-based biosensor showed a 27% decrease in the BRET ratio in the presence of saturating (1 mM) lactose. Using a 5 min assay, the half maximal effective concentration (EC50) for lactose in phosphate-buffered saline (PBS) was 12 µM. The biosensor was 200 times more sensitive to lactose than to glucose or galactose. Sensitivity and selectivity were not significantly affected by the presence of 10% (v/v) dialyzed milk. The biosensor is suitable for direct determination of residual lactose in lactase-treated milk, with a limit of detection of 0.2 µM, 100 times below the most stringent lactose-free standard and without the need to remove fat or protein from the sample.

Interaction via the N terminus of the type IV secretion system (T4SS) protein VirB6 with VirB10 is required for VirB2 and VirB5 incorporation into T-pili and for T4SS function.

Many bacterial pathogens employ multicomponent protein complexes such as type IV secretion systems (T4SSs) to transfer virulence factors into host cells. Here we studied the interaction between two essential T4SS components: the very hydrophobic inner membrane protein VirB6, which may be a component of the translocation channel, and VirB10, which links the inner and outer bacterial membranes. To map the interaction site between these two T4SS components, we conducted alanine scanning and deleted six-amino acid stretches from the N-terminal periplasmic domain of VirB6 from Brucella suis Using the bacterial two-hybrid system to analyze the effects of these alterations on the VirB6-VirB10 interaction, we identified the amino acid regions 16-21 and 28-33 and Leu-18 in VirB6 as being required for this interaction. SDS-PAGE coupled with Western blotting of cell lysates and native PAGE of detergent-extracted membrane proteins revealed that the corresponding VirB6 residues in Agrobacterium tumefaciens (Phe-20 and amino acids 18-23 and 30-35) modulate the stability of both VirB6 and VirB5. However, the results from immuno-EM and super-resolution microscopy suggested that these regions and residues are not required for membrane association or for polar localization of VirB6. The six-amino acid deletions in the N terminus of VirB6 abolished pilus formation and virulence of A. tumefaciens, and the corresponding deletions in the VirB6 homolog TraD from the plasmid pKM101-T4SS abrogated plasmid transfer. Our results indicate that specific residues of the VirB6 N-terminal domain are required for VirB6 stabilization, its interaction with VirB10, and the incorporation of VirB2 and VirB5 into T-pili.