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Diarrheal diseases remain a major public health concern worldwide. Many of the causative bacterial pathogens that cause these diseases have a specialized protein complex, the type III secretion system (T3SS), which delivers effector proteins directly into host cells. These effectors manipulate host cell processes for the benefit of the infecting bacteria. The T3SS structure resembles a syringe anchored within the bacterial membrane, projecting toward the host cell membrane. The entry port of the T3SS substrates, called the export apparatus, is formed by five integral membrane proteins. Among the export apparatus proteins, EscV is the largest, and as it forms a nonamer, it constitutes the largest portion of the export apparatus complex. While there are considerable data on the soluble cytoplasmic domain of EscV, our knowledge of its membrane-associated section and its transmembrane domains (TMDs) is still very limited. In this study, using an isolated genetic reporter system, we found that TMD5 and TMD6 of EscV mediate strong self-oligomerization. Substituting these TMDs within the full-length protein with a random hydrophobic sequence resulted in a complete loss of function of the T3SS, further suggesting that the EscV TMD5 and TMD6 sequences have a functional role in addition to their structural role as membrane anchors. As we observed only mild reduction in the ability of the TMD-exchanged variants to integrate into the full or intermediate T3SS complexes, we concluded that EscV TMD5 and TMD6 are not crucial for the global assembly or stability of the T3SS complex but are rather involved in promoting the necessary TMD–TMD interactions within the complex and the overall TMD orientation to allow channel opening for the entry of T3SS substrates. [ABSTRACT FROM AUTHOR]

Many gram-negative pathogens utilize a protein complex, termed the type III secretion system (T3SS), to inject virulence factors from their cytoplasm directly into the host cell. An export apparatus that is formed by five putative integral membrane proteins (SctR/S/T/U/V), resides at the center of the T3SS complex. In this study, we characterized the smallest export apparatus protein, SctS, which contains two putative transmembrane domains (PTMD) that dynamically extract from the inner membrane and adopt a helix-turn-helix structure upon assembly of the T3SS. Replacement of each SctS PTMD with an alternative hydrophobic sequence resulted in abolishment of the T3SS activity, yet SctS self- and hetero-interactions as well as the overall assembly of the T3SS complex were unaffected. Our findings suggest that SctS PTMDs are not crucial for the interactions or the assembly of the T3SS base complex but rather that they are involved in adjusting the orientation of the export apparatus relative to additional T3SS sub-structures, such as the cytoplasmic- and the inner-membrane rings. This ensures the fittings between the dynamic and static components of the T3SS and supports the functionality of the T3SS complex. [ABSTRACT FROM AUTHOR]

Catabolite repression is a mechanism that enables bacteria to control carbon utilization. As part of this global regulatory network, components of the phosphoenolpyruvate:carbohydrate phosphotransferase system inhibit the uptake of less favorable sugars when a preferred carbon source such as glucose is available. This process is termed inducer exclusion. In bacteria belonging to the phylum Firmicutes, HPr, phosphorylated at serine 46 (P-Ser46-HPr) is the key player but its mode of action is elusive. To address this question at the level of purified protein components, we have chosen a homolog of the Escherichia coli maltose/maltodextrin ATP-binding cassette transporter from Lactobacillus casei (MalE1-MalF1G1K12) as a model system. We show that the solute binding protein, MalE1, binds linear and cyclic maltodextrins but not maltose. Crystal structures of MalE1 complexed with these sugars provide a clue why maltose is not a substrate. P-Ser46-HPr inhibited MalE1/maltotetraose-stimulated ATPase activity of the transporter incorporated in proteoliposomes. Furthermore, cross-linking experiments revealed that P-Ser46-HPr contacts the nucleotide-binding subunit, MalK1, in proximity to the Walker A motif. However, P-Ser46-HPr did not block binding of ATP to MalK1. Together, our findings provide first biochemical evidence that P-Ser-HPr arrests the transport cycle by preventing ATP hydrolysis at the MalK1 subunits of the transporter. [ABSTRACT FROM AUTHOR]

This study was conducted to examine the effects of dietary valine on the physical and flavor characteristics, fatty acid (FA) profile, antioxidant status and Nrf2-dependent antioxidant enzyme gene expression in the muscle of young grass carp (Ctenopharyngodon idella) fed increasing levels of valine (4.3, 8.0, 10.6, 13.1, 16.9 and 19.1 g/kg) for 8 weeks. Compared with the control group, the group fed valine showed improved physical characteristics of fish fillets (increased relative shear force, hydroxyproline, protein and lipid levels and decreased cathepsin B and L activities, as well as cooking loss, were observed). Moreover, valine improved the flavor of young grass carp fillets by increasing the amino acid (AA) concentration in fish muscle (increased aspartic acid, threonine, glutamine, cystine, methionine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine and valine concentrations were observed). Additionally, optimal valine supplementation increased the potential health benefits to humans by decreasing the saturated FA (C15:0 and C16:0) concentration and increasing the unsaturated FA (monounsaturated FAs (MUFAs), such as C16:1, C18:1c+t and C20:1, and polyunsaturated FAs (PUFAs), such as C18:3n-3, C20:2 and C22:6) concentration. In addition, the reduced glutathione (GSH) content and the activities of Cu/Zn superoxide dismutase (SOD1), catalase (CAT) and Selenium-dependent glutathione peroxydase (Se-GPx) increased under valine supplementation (P < 0.05). Furthermore, the SOD1, CAT and Se-GPx mRNA levels increased with dietary valine levels, possibly due to the up-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the down-regulation of Kelch-like-ECH-associated protein 1 (Keap1) in muscle (P < 0.05). In conclusion, valine improved the physical and flavor characteristics, FA profile, and antioxidant status and regulated the expression of the antioxidant enzyme genes Nrf2, Keap1, TOR and S6K1 in fish fillets. [ABSTRACT FROM AUTHOR]

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems. [ABSTRACT FROM AUTHOR]

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which b-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to b-lactam even though they do not encode allosteric enzymes. These 'phenotypic switch' genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo. [ABSTRACT FROM AUTHOR]

In order to fulfill their function, membrane transport proteins have to cycle through a number of conformational and/or energetic states. Thus, understanding the role of conformational dynamics seems to be the key for elucidation of the functional mechanism of these proteins. However, membrane proteins in general are often difficult to express heterologously and in sufficient amounts for structural studies. It is especially challenging to trap a stable energy minimum, e.g., for crystallographic analysis. Furthermore, crystallization is often only possible by subjecting the protein to conditions that do not resemble its native environment and crystals can only be snapshots of selected conformational states. Nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy are complementary methods that offer unique possibilities for studying membrane proteins in their natural membrane environment and for investigating functional conformational changes, lipid interactions, substrate-lipid and substrate-protein interactions, oligomerization states and overall dynamics of membrane transporters. Here, we review recent progress in the field including studies from primary and secondary active transporters. [ABSTRACT FROM AUTHOR]

To examine the relationship between folding and aggregation in the periplasm of Escherichia coli, we have analysed the cellular fates of exported proteins fused to either the wild-type maltose-binding protein (MalE) or the aggregation-prone variant MalE31. The propensity of fusion proteins to aggregate in the periplasm was determined by the intrinsic folding characteristics of the upstream protein. When β-lactamase or alkaline phosphatase was linked to the C-terminus of MalE31, the resultant fusion proteins accumulated in an insoluble form, but retained their catalytic activity. In addition, these protein aggregates induced an extracytoplasmic stress response, similar to unfused MalE31. However, using a fluorescent substrate, we found that alkaline phosphatase activity was present inside periplasmic aggregates. These results suggest that periplasmic inclusion body formation may result in intermolecular interactions between participating proteins without loss of function of the fused enzymes. [ABSTRACT FROM AUTHOR]

Thermodynamic stability is fundamental to the biology of proteins. Information on protein stability is essential for studying protein structure and folding and can also be used indirectly to monitor protein-ligand or protein-protein interactions. While clearly valuable, the experimental determination of a protein's stability typically requires biophysical instrumentation and substantial quantities of purified protein, which has limited the use of this technique as a general laboratory method. We report here a simple new method for determining protein stability by using pulse proteolysis with varying concentrations of denaturant. Pulse proteolysis is designed to digest only the unfolded proteins in an equilibrium mixture of folded and unfolded proteins that relaxes on a time scale longer than the proteolytic pulse. We used this method to study the stabilities of Escherichia coli ribonuclease H and its variants, both in purified form and directly from cell lysates. The ΔGunf° values obtained by this technique were in agreement with those determined by traditional methods. We also successfully used this method to monitor the binding of maltose-binding protein to maltose, as well as to rapidly screen cognate ligands for this protein. The simplicity of pulse proteolysis suggests that it is an excellent strategy for the high-throughput determination of protein stability in protein engineering and drug discovery applications. [ABSTRACT FROM AUTHOR]

Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s-1) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100-150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryophotolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions. [ABSTRACT FROM AUTHOR]

The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel β-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, ΔL4, ΔL5, ΔL6, and double-loop deletions, ΔL4 + ΔL5 and ΔL5 + ΔL6, abolished maltoporin functions, but not the double deletion ΔL4 + ΔL6 and the triple deletion ΔL4 + ΔL5 + ΔL6. While deletion of the central variable portion of loop L9 (ΔL9v) affected maltoporin function only moderately, the combination of ΔL9v with the double deletion of loops L4 and L6 (triple deletion ΔL4 + ΔL6 + ΔL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBΔL9v), of two loops L4 and L6 (LamBΔL4 + ΔL6), of three loops L4, L5 and L6 (LamBΔL4 + ΔL5 + ΔL6) or of L4, L6 and L9v (LamBΔL4 + ΔL6 + ΔL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBΔL4) or L6 (LamBΔL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column,... [ABSTRACT FROM AUTHOR]

The transport of solutes by bacteria has been studied for about thirty years. Early experiments on amino acid entry and galactoside accumulation provided concrete evidence that bacteria possessed specific transport systems and that these were subject to regulation. Since than a large number of transport systems have been discovered and studied extensively. Many of these use entirely different strategies for capturing or accumulating substrates. This diversity reflects variation in the availability of nutrients an ions in the different environments tolerated and inhabited by microorganisms. Examination of a few bacterial transport systems provides an opportunity to gain insight into a wide range of topics in the area of membrane transport. These include: the identification of carrier proteins and their arrangement in the membrane, the regulation of transport protein synthesis by environmental factors, and the localization of transport proteins to their extracytoplasmic destinations. It has been possible to construct a number of bacterial strains in which the gene (lacZ) which codes for the cytoplasmic enzyme beta-galactosidase is fused to genes which code for transport proteins. The following article is intended to illustrate how these gene fusions have been used to study the regulation and structure of transport proteins in Escherichia coli. [ABSTRACT FROM AUTHOR]

The outer membrane of Vibrio cholerae contains a maltose-inducible major protein, OmpS (43 kDa), that is common to different isolates. Nucleotide sequence analysis of the corresponding structural gene, ompS, revealed an open reading frame encoding a 412-amino-acid polypeptide. The amino acid sequence of OmpS is similar to that of LamB, the Escherichia coli maltoporin, and to ScrY of Kiebsiella pneumoniae, although the antigenic determinants of these proteins are different. The cloned ompS gene complemented an ompS mutation of V. cholerae and the corresponding polypeptide could function as a maltoporin in a LamB- mutant of E. coli The promoter region of ompS is highly homologous to the malK-lamB promoter of E. coli and the ompS gene is controlled by MalT in E. coli This indicates that the same kind of regulatory mechanism is used to activate the ompS expression in V. cholerae and malK-lamB expression in E. coli An ompS-lacZ transcriptional fusion was used to demonstrate a dual control in ompS expression; the ompS gene is responsive to the inducers maltose and trehalose but in their absence it is also expressed in response to growth-phase. These different modes of induction might be of importance during different stages of V. cholerae infection. [ABSTRACT FROM AUTHOR]

A membrane-bound fraction of polysomes of Escherichia coli has been isolated after lysis of cells without the use of lysozyme. Protein-synthesis studies in vitro show that membrane-bound and free polysomes are different in the following respects. 1. Membrane-bound polysomes synthesize proteins which are exported from the cell. The products include proteins of the outer membrane and secreted periplasmic protein, the maltose-binding protein. 2. The major product synthesized by free polysomes is elongation factor Tu, a soluble cytoplasmic protein. 3. The activity of membrane-bound polysomes in vitro is more resistant to puromycin than is the activity of free polysomes. In addition, the mRNA associated with membrane-bound polysomes is more stable than the bulk of cellular mRNA as revealed by studies with rifampicin. [ABSTRACT FROM AUTHOR]

The kinetic parameters for the maltose transport system in Escherichia coli K12 were determined with maltose and maltotriose as substrates. The system exhibits an apparent Km of 1μM for maltose and 2 μM for maltotriose. The V of entry was determined as 2.0 and 1.1 mol substrate/min per 108 cells. Mutations in lamB, the structural gene for the receptor protein of phage λ, increased the Km for maltose transport by a factor of 100–500 without influencing the maximal rate of transport. Maltotriose is no longer transported in these lamB mutants. The maltose-binding protein, an essential component of the maltose transport system, was found .to exhibit substrate-dependent fluorescence quenching. This phenomenon was used to determine dissociation constants and to estimate the rate of ligand dissociation. A Kd of 1 μM for maltose and of 0.16 μM for maltotriose was found. From the comparison of the kinetic parameters of transport of maltose and maltotriose in wild-type and λ-resistant mutants with the binding constants for both sugars to purified maltose-binding protein, we conclude that the λ receptor facilitates the diffusion of maltose and maltodextrins through the outer membrane. [ABSTRACT FROM AUTHOR]

The biphasic binding properties of the galactose-binding and maltose-binding proteins of Escherichia coli may be important in the functioning of these proteins as recognition components of chemoreceptors. However, Richarme and Kepes [Eur. J. Biochem. 45, 127-133 (1974)] have suggested that the biphasic binding curie of the galactose, binding protein may be the result of isotopic dilution, during equilibrium dialysis, by unlabeled ligand retained by the binding throughout purification. Here the binding of maltose to maltose-binding protein which has never previously been exposed to the sugar ('virgin' binding protein) is shown to be biphasic. This implies that the unusual binding properties are attributable to the maltose-binding protein itself. [ABSTRACT FROM AUTHOR]

ScrY, an outer membrane channel of enteric Gram-negative bacteria, which confers to the bacteria the rapid uptake of sucrose through the outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated in the open and in the carbohydrate-induced closed state of the channel. The open state of the channel exhibited up to about 200 Hz 1/f-noise with a rather small spectral density. Upon addition of carbohydrates to the aqueous phase the current through the ScrY channels decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the carbohydrates with the binding site inside the channel and its reversible block. The frequency dependence of the spectral density was of the Lorentzian type but very often two Lorentzians were observed, from which the slow one may not be related to carbohydrate binding. Analysis of the power density spectra of the second Lorentzian using a previously proposed simple model of carbohydrate binding allowed the evaluation of the on- and the off-rate constants for the carbohydrate association with the binding site inside the ScrY channel and of a mutant (ScrYDelta3-72), in which 70 amino acids at the N-terminus are deleted. The binding of carbohydrates to ScrY was compared to those of the closely related maltoporin channels of Escherichia coli and Salmonella typhimurium by assuming that only the time constant and spectral density of the high frequency Lorentzian is related to carbohydrate transport. [ABSTRACT FROM AUTHOR]

In Escherichia coli K12 the product of gene lamB is an outer membrane protein involved in the transport of maltose and maltodextrins and serving as a receptor for several bacteriophages including λ. About 30 to 40% of this protein can be recovered associated to peptidoglycan when the cells are dissolved in sodium dodecyl sulfate in the presence of 2 mM Mg2+ ions. The bound protein can then be quantitatively eluted from peptidoglycan by incubating the complex in Triton X-100 and EDTA, or sodium dodecyl sulfate and NaCl. The protein eluted in such ways is still totally active in its phage-neutralizing activity. Two other outer membrane proteins known to behave similarly to the lamB protein are proteins Ia and lb. However the binding of these proteins to peptidoglycan appears tighter, in several respects, than that of the lamB protein. The lamB protein may span the outer membrane since it appears to interact with the peptidoglycan on the inner side of this membrane while it is known to be accessible to both phages and antibodies at the cell surface. [ABSTRACT FROM AUTHOR]