Purification of soluble and active RaxH, a transmembrane histidine protein kinase from Xanthomonas oryzae pv. oryzae required for AvrXa21 activity

The RaxHR two-component regulatory system (TCS) of the rice pathogen Xanthomonas oryzae pv. oryzae is required for AvrXa21 activity. RaxH is a typical transmembrane histidine protein kinase (HK), whereas RaxR is its concomitant response regulator (RR). Here, we report the isolation of soluble, active amounts of recombinant His-tagged full-length RaxH and RaxR following growth of Escherichia coli over-expressing strains in the presence of sorbitol and glycine betaine. Full-length His-RaxH showed similar autophosphorylation activities to that of a truncated version of the protein (His-t-RaxH), lacking the N-terminal transmembrane region. Transphosphorylation assays revealed that only full-length RaxH was able to induce phosphorylation of His-RaxR, indicating that the N-terminal region of RaxH may be required for transphosphorylation of RaxR. Using site-directed mutagenesis we also demonstrated that residues histidine 222 in RaxH and aspartate 51 in RaxR are essential for phosphorylation activities of these proteins. Utilization of compatible solutes may be widely applied for purification of soluble, active recombinant transmembrane proteins, and in particular for purification of transmembrane HKs.     

Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches

Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345). This property limits genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies.     

The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein

Abstract The phosphoglycerate kinase ( pgk ), triosephosphate isomerase ( tpi ), and enolase ( eno ) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli . In high copy number, the pgk gene complemented an E. coli pgk ⁻ strain. In T. neapolitana , the pgk and tpi genes appear to be fused and eno is near those genes. Like T. maritima , T. neapolitana produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity. Unlike T. maritima , which forms only a 70-kDa fusion protein, T. neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein. These isozymes are present in both T. neapolitana cells and in E. coli cells expressing T. neapolitana genes.     

Solution structure of the bacterial chemotaxis adaptor protein CheW from Escherichia coli

The bacterial chemotaxis adaptor protein CheW physically links the chemoreceptors (MCPs) and the histidine kinase CheA. Extensive investigations using bacterium Escherichia coli have established the central role of CheW in the MCP-modulated activation of CheA. Here we report the solution structure of CheW from E. coli determined by NMR spectroscopy. The results show that E. coli CheW shares an overall fold with previously reported structure of CheW from Thermotoga maritima, whereas local conformational deviations are observed. In particular, the C-terminal alpha-helix is considerably longer in E. coli CheW and appears to shrink the active binding pocket with CheA. Our study provides the structural basis for further investigations in E. coli chemotaxis.     

A truncated H-NS-like protein from enteropathogenic Escherichia coli acts as an H-NS antagonist

The H-NS nucleoid-associated protein of Escherichia coli is the founder member of a widespread family of gene regulatory proteins which have a bipartite structure, consisting of an N-terminal coiled-coil oligomerization domain and a C-terminal DNA-binding domain. Here we characterize a family of naturally occurring truncated H-NS derivatives lacking the DNA-binding domain, which we term the H-NST family. H-NST proteins are found in large genomic islands in pathogenic E. coli strains, which are absent from the corresponding positions in the E. coli K-12 genome. Detailed analysis of the H-NST proteins from enteropathogenic E. coli (EPEC) and uropathogenic E. coli (UPEC) shows that the EPEC protein (H-NST(EPEC)) has a potent anti-H-NS function at the classical H-NS-repressed operon proU. This correlates with the ability of H-NST(EPEC) to co-purify with H-NS in vitro, and can be abolished by a mutation of leucine 30 to proline which is predicted to prevent the N-terminal region from forming a coiled-coil structure. In contrast, despite being 90% identical to H-NST(EPEC) at the protein level, the UPEC homologue (H-NST(UPEC)) has only a weak anti-H-NS activity, correlating with a much-reduced ability to interact with H-NS during column chromatography. A single amino acid difference at residue 16 appears to account for these different properties. The hnsT(EPEC) gene is transcribed monocistronically and expressed throughout the exponential growth phase in DMEM medium. Our data suggest that a truncated derivative of H-NS encoded by an ancestral mobile DNA element can interact with the endogenous H-NS regulatory network of a bacterial pathogen.     

Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima.

An expressed sequence tag homologous to cheA was previously isolated by random sequencing of Thermotoga maritima cDNA clones (C. W. Kim, P. Markiewicz, J. J. Lee, C. F. Schierle, and J. H. Miller, J. Mol. Biol. 231: 960-981, 1993). Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T. maritima lambda library by using PCR. Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced. The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY. In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function. Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors. Upon induction, both proteins were synthesized at high levels. T. maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E. coli protein by heat treatment at 80 degrees C for 10 min. CheY prepared in this way was shown to be active by the demonstration of Mg(2+)-dependent autophosphorylation with [32P]acetyl phosphate. In E. coli, CheW mediates the physical coupling of the receptors to the kinase CheA. The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.     

Oligomeric state and mode of self-association of Thermotoga maritima ribosomal stalk protein L12 in solution

The "stalk" of the prokaryotic 50S ribosomal subunit is comprised of four copies of the protein L7/L12. In Escherichia coli, L7/L12 is a dimeric protein at micromolar concentrations, which is able to undergo rapid subunit exchange. A recent structural study indicated a tetrameric arrangement of the L12 proteins isolated from Thermotoga maritima, in which the proteins engaged in two different dimerization modes. In one mode, the two monomers of L12 form a tight symmetric and parallel dimer held together by a four-helix bundle, which encompasses the hinge region between the N- and C-terminal domains. In the other mode, the two monomers bind through their N-terminal region in an antiparallel configuration, in which one monomer comprises an alpha-helical hinge and the other monomer adopts an elongated shape with an unfolded hinge region. Presently, it is unclear which dimer contact prevails in solution and on the ribosome. Using cysteine mutants of T. maritima labeled with fluorescent probes, we investigated the mode of interactions between L12 subunits. Data from Forster resonance energy transfer experiments support a dimerization of L12 in solution, in which two monomers bind through their N-terminal region in an antiparallel configuration. We also demonstrate that the rate of subunit exchange in T. maritima L12 is significantly slower at 25 degrees C than that in the E. coli system. The exchange rate increases with increasing temperature and approaches the one observed for the E. coli system at 50 degrees C. Possible factors responsible for this difference are discussed.     

Membrane topology of the DrrB protein of the doxorubicin transporter of Streptomyces peucetius

Daunorubicin and doxorubicin, two commonly used anticancer agents, are produced by the soil bacterium Streptomyces peucetius. Self-resistance to these antibiotics in S. peucetius is conferred by the drrAB locus that codes for two proteins, DrrA and DrrB. DrrA is an ATP-binding protein. It belongs to the ABC family of transporters and shares sequence and functional similarities with P-glycoprotein of cancer cells. DrrB is an integral membrane protein that might function as a transporter for the efflux of daunorubicin and doxorubicin. Together, DrrA and DrrB are believed to form an ATP-driven pump for the efflux of these drugs. The drrAB locus has been cloned, and the two proteins have been expressed in a functional form in Escherichia coli. A topological analysis of the DrrB protein was performed using gene fusion methodology. Random and site-directed fusions of the drrB gene to lacZ, phoA, or gfp reporter genes were created. Based on the fusion data, a topological model of the DrrB protein is proposed in which the protein has eight membrane-spanning domains with both the N terminus and the C terminus in the cytoplasm.     

Expression of polyoma early gene products in E. coli.

The three products of the early region of polyoma virus have been cloned for expression in E. coli using the Tac promoter. Although the identical promoter and ribosome binding site are used in each final construction, the observed level of protein expression is different for each protein. While plasmids expressing wild type T antigens as well as a plasmid expressing the truncated Py-1387T middle T antigen lacking the membrane-anchoring sequence give rise to synthesis of proteins readily detectible by 35S-methionine labeling and immunoprecipitation, only small T and the middle T of Py-1387T are made in amounts sufficient for ready detection in total cell protein. Unlike middle T expressed in animal cells, middle T produced in E. coli is not detectibly phosphorylated. Further, the E. coli protein lacks tyrosine kinase activity.     

Coexpression of the long and short forms of CheA, the chemotaxis histidine kinase, by members of the family Enterobacteriaceae.

CheA is the histidine protein kinase of a two-component signal transduction system required for bacterial chemotaxis. Motile cells of the enteric species Escherichia coli and Salmonella typhimurium synthesize two forms of CheA by utilizing in-frame initiation sites within the gene cheA. The full-length protein, CheAL, plays an essential role in the chemotactic signaling pathway. In contrast, the function of the short form, CheAs, remains elusive. Although CheAs lacks the histidine residue that becomes phosphorylated in CheAL, it exhibits both kinase activity and the ability to interact with and enhance the activity of CheZ, a chemotaxis protein that accelerates dephosphorylation of the two-component response regulator CheY. To determine whether other members of the family Enterobacteriaceae express CheAs and CheZ, we analyzed immunoblots of proteins from clinical isolates of a variety of enteric species. All motile, chemotactic isolates that we tested coexpressed CheAL, CheAs, and CheZ. The only exceptions were closely related plant pathogens of the genus Erwinia, which expressed CheAL and CheZ but not CheAs. We also analyzed nucleotide sequences of the cheA loci from isolates of Serratia marcescens and Enterobacter cloacae, demonstrating the presence of in-frame translation initiation sites similar to those observed in the cheA loci of E. coli and S. typhimurium. Since coexpression of CheAs and CheZ appears to be limited to motile, chemotactic enteric bacteria, we propose that CheAs may play an important role in chemotactic responses in some environmental niches encountered by enteric species.     

Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.     

A NusG-like protein from Thermotoga maritima binds to DNA and RNA.

The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity. Purified T. maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity. The complexes formed between nucleic acid and T. maritima NusG were unable to penetrate a polyacrylamide or agarose gel. The affinity of the protein for DNA was highest in buffers containing about 50 mM salt. The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T. maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA. The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA. Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis. Neither the wild-type T. maritima nusG gene nor a deletion derivative more similar to the E. coli gene was able to substitute for the essential E. coli nusG. Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T. maritima NusG, and the other has only the sequences present in NusG homologs from E. coli and other eubacteria. Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T. maritima NusG.     

Truncated forms of Escherichia coli lactose permease: models for study of biosynthesis and membrane insertion.

Using in vitro DNA manipulations, we constructed different lacY alleles encoding mutant proteins of the Escherichia coli lactose carrier. With respect to structural models developed for lactose permease, the truncated polypeptides represent model systems containing approximately one, two, four, and five of the N-terminal membrane-spanning alpha-helices. In addition, a protein carrying a deletion of predicted helices 3 and 4 was obtained. The different proteins were radiolabeled in plasmid-bearing E. coli minicells and were found to be stably integrated into the lipid bilayer. The truncated polypeptides of 50, 71, 143, and 174 N-terminal amino acid residues resembled the wild-type protein in their solubilization characteristics, whereas the mutant protein carrying an internal deletion of amino acid residues 72 to 142 of the lactose carrier behaved differently. Minicell membrane vesicles containing truncated proteins comprising amino acid residues 1 to 143 or 1 to 174 were subjected to limited proteolysis. Upon digestion with proteases of different specificities, the same characteristic fragment that was also produced from the membrane-associated wild-type protein was found to accumulate under these conditions. It has previously been shown to contain the intact N terminus of lactose permease. This supports the idea of an independent folding and membrane insertion of this segment even in the absence of the C-terminal part of the molecule. The results suggest that the N-terminal region of the lactose permease represents a well-defined structural domain.     

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.     

Hyperthermophilic topoisomerase I from Thermotoga maritima. A very efficient enzyme that functions independently of zinc binding

Topoisomerases, by controlling DNA supercoiling state, are key enzymes for adaptation to high temperatures in thermophilic organisms. We focus here on the topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima (optimal growth temperature, 80 degrees C). To determine the properties of the enzyme compared with those of its mesophilic homologs, we overexpressed T. maritima topoisomerase I in Escherichia coli and purified it to near homogeneity. We show that T. maritima topoisomerase I exhibits a very high DNA relaxing activity. Mapping of the cleavage sites on a variety of single-stranded oligonucleotides indicates a strong preference for a cytosine at position -4 of the cleavage, a property shared by E. coli topoisomerase I and archaeal reverse gyrases. As expected, the mutation of the putative active site Tyr 288 to Phe led to a totally inactive protein. To investigate the role of the unique zinc motif (Cys-X-Cys-X(16)-Cys-X-Cys) present in T. maritima topoisomerase I, experiments have been performed with the protein mutated on the tetracysteine motif. Strikingly, the results show that zinc binding is not required for DNA relaxation activity, contrary to the E. coli enzyme. Furthermore, neither thermostability nor cleavage specificity is altered in this mutant. This finding opens the question of the role of the zinc-binding motif in T. maritima topoisomerase I and suggests that this hyperthermophilic topoisomerase possesses a different mechanism from its mesophilic homolog.     

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.     

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.     

Cloning, sequencing, and expression of ruvB and characterization of RuvB proteins from two distantly related thermophilic eubacteria.

The ruvB genes of the highly divergent thermophilic eubacteria Thermus thermophilus and Thermotoga maritima were cloned, sequenced, and expressed in Escherichia coli. Both thermostable RuvB proteins were purified to homogeneity. Like E. coli RuvB protein, both purified thermostable RuvB proteins showed strong double-stranded DNA-dependent ATPase activity at their temperature optima (> or = 70 degrees C). In the absence of ATP, T. thermophilus RuvB protein bound to linear double-stranded DNA with a preference for the ends. Addition of ATP or gamma-S-ATP destabilized the T. thermophilus RuvB-DNA complexes. Both thermostable RuvB proteins displayed helicase activity on supercoiled DNA. Expression of thermostable T. thermophilus RuvB protein in the E. coli ruvB recG mutant strain N3395 partially complemented the UV-sensitive phenotype, suggesting that T. thermophilus RuvB protein has a function similar to that of E. coli RuvB in vivo.