Isolation and Purification of Two Novel Streptomycete RNase Inhibitors, SaI14 and SaI20, and Cloning, Sequencing, and Expression in Escherichia coli of the Gene Coding for SaI14

Two new RNase inhibitors, SaI14 (M_r, ~14,000) and SaI20 (M_r, ~20,000), were isolated and purified from a Streptomyces aureofaciens strain. The gene sai14, coding for SaI14 protein, was cloned and expressed in Escherichia coli. The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor from Bacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Letter: Crystallographic data of an L-arabinose-binding protein from Escherichia coli

Single crystals of an l-arabinose-binding protein from Escherichia coli B/r suitable for a high-resolution structural analysis have been obtained. An X-ray examination of the orthorhombic crystals shows the space group is P2₁2₁2₁, with unit cell dimensions: $\text{a = 55.3}\text{A}\text{?}\text{, b = 71.4}\text{A}\text{?}\text{and}\text{c = 77.5}\text{A}\text{?}$. The asymmetric unit contains one protein molecule with a molecular weight of 38,000.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

Cloning and sequencing of the gene for the DNA-binding 17 K protein of Escherichia coli

The skp gene encoding the 17 K protein, a basic DNA-binding nucleoid-associated protein of Escherichia coli, was cloned as part of a 2.3-kb genomic fragment. The gene was sequenced and a polypeptide of 161 amino acids (aa) was deduced from the nucleotide sequence. The primary translation product was processed by cutting off the N-terminal 20 aa residues, yielding a mature polypeptide of 141 aa. The M_r of the mature polypeptide was 15674. An E. coli transformant containing the skp gene on the plasmid pGAH317 was shown to overproduce the gene product some 20-fold.     

Isolation of a mannose-specific lectin from Escherichia coli and its role in the adherence of the bacteria to epithelial cells.

A high molecular weight protein aggregate, which agglutinates yeast cells, human epithelial cells and mouse lymphocytes, was isolated from extracts of Escherichia coli by differential centrifugation and gel filtration. The agglutination is specifically inhibited by d-mannose and its derivatives, the best inhibitor being p-nitrophenyl α-d-mannoside. Sodium dodecyl sulfate gel electrophoresis showed that the lectin consists of protein subunits with identical M_r of ～36500. The amino acid composition of the purified lectin is different from that reported for the type I pili protein, the K99 antigen and the major outer membrane protein Ia of E. coli. The protein appears to be located on the bacterial surface, and is probably involved in the mannose-specific adherence of E. coli to eukaryotic cells.     

Purification and properties of two truncated endoglucanases produced in Escherichia coli harbouring Clostridium cellulolyticum endoglucanase gene celCCD

The endoglucanase gene, celCCD, of Clostridium cellulolyticum has been expressed in Escherichia coli. Multiple active polypeptides were detected in the E. coli cells. The relative molecular mass (M_r) of two major active polypeptides were 56 000 (D56) and 38 000 (D38), which were smaller than the deduced M_r of the mature protein (63 401). D56 and D38 were purified from the periplasmic fraction. The N-terminal sequences of the two purified polypeptides were identical to that of the mature endoglucanase (Ala-Ile-Asn-Ser-Gln-Asp-Met-Val---) deduced from the nucleotide sequence. These data indicated that these polypeptides were produced by processing the original mature protein in the C-terminal region. The enzymatic properties of these two polypeptides were very similar, except that the specific activity of D38 was 2–3.5-fold higher than that of D56, and D38 was more heat stable than D56. Correspondence to: T. Kodama     

Cloning and expression of theClostridium thermocellum celS gene inEscherichia coli

Clostridium thermocellum ATCC 27405 produces an extremely complicated multi-component cellulase aggregate (cellulosome) highly active on crystalline cellulose. From the cellulosome, two subunits, CelS (or S _s ;M _r = 82 000) and CelL (or S _l , CipA;M _r = 250 000), have been identified as essential for crystalline cellulose degradation [Wu et al. (1988) Biochemistry 27:1703]. We have determined the DNA sequence of thecelS gene from four cloned DNA fragments encompassing this gene [Wang et al. (1993) J Bacteriol 175:1293]. To express the entirecelS gene inEscherichia coli, thecelS structural gene was amplified by the polymerase chain reaction (PCR) employing the PCR primers corresponding to sequences flanking the desired gene. This PCR product (2.1 x 10³ bases; 2.1 kb) was cloned into anE. coli expression vector pRSET B. Subsequent expression of the cloned gene resulted in a fusion protein (rCelS;M _r = 86 000) as inclusion bodies. The rCelS protein was recognized specifically by an anti-CelS antiserum in a Western blot analysis. The inclusion bodies were purified and solubilized in 5m urea. The refolded rCelS produced very little reducing sugar from carboxymethylcellulose. However, it showed a higher activity on the crystalline cellulose (Avicel) and an even higher activity on phosphoricacid-swollen Avicel. These results indicate that the CelS is an exoglucanase.     

A protein specific to mitochondria from S-type male-sterile cytoplasm of maize is encoded by an episomal DNA

Mitochondria of S-type cytoplasmic male sterile maize contain two linear double-stranded DNA molecules, S1 and S2. Two open reading frames (ORF1 and ORF2) are present in S2 DNA. Fragments from ORF1 were inserted into plasmids to achieve expression in Escherichia coli. Cells transformed with recombinant plasmids produced mRNA which hybridized with ORF1 and corresponding polypeptides were synthesized by in vivo and in vitro systems. Antiserum against a lacZ/S2 fusion protein precipitated the anticipated polypeptides from transformed E. coli cells and was therefore used to detect homologous peptide sequences in protein preparations of mitochondria from different maize cytoplasms. The antiserum detected a protein of 125 000 M_r present in mitochondria from male sterile B73S but absent from the fertile B73N cytoplasm.     

Purification and properties of Escherichia coli coenzyme A-transferase

The acetyl-CoA:acetoacetate-CoA-transferase has been purified 36-fold to homogeneity from an acetoacetate degradation operon (ato) constitutive mutant of Escherichia coli. The enzyme has the following physical properties: Stokes radius, 40.5 Å; diffusion coefficient (D₂₀,w), 5.32 × 10^?7 cm s^?1; sedimentation coefficient (s₂₀,w), 5.38S; molecular weight, 97,000 and a frictional ratio ($\text{f}\text{f}{}_0$) of 1.35. The enzyme is composed of two α subunits (M_r = 26,000) and two β subunits (M_r = 23,000). E. coli CoA-transferase contains six cysteine residues per mole of enzyme and no disulfide bonds. The native transferase reacts with 4 mol of p-chloromercuribenzoate per 97,000 g of enzyme. Two cysteine residues react rapidly with p-chloromercuribenzoate resulting in an 85% inactivation of enzyme activity. The reactivity of these two residues is enhanced at least fivefold in the presence of acetyl-CoA. Acetoacetate has no effect on the rate of reaction of p-chloromercuribenzoate with the enzyme. E. coli CoA-transferase is partially inactivated by acyl-CoA substrates in the absence of carboxylic acid substrates, presumably as the result of a metal-catalyzed acylation of the ?-amino group of a lysine residue near the active site. The enzyme utilizes a variety of short chain acyl-CoA and carboxylic acid substrates but exhibits maximal activity with normal and 3-keto substrates.     

Predominant end-products of prophage Mu DNA transposition during the lytic cycle are replicon fusions

The structure of l-arabinose-binding protein (M_r 33, 100), an essential component of the osmotic shock-sensitive, high-affinity l-arabinose transport system in Escherichia coli, has been determined at 2.4 Å resolution. The phases were solved by the method of multiple isomorphous replacement, using four derivatives, p-chloromercuribenzenesulfonate and CdI₂ (data to 2.4 Å resolution), and p-chloromercurinitrophenol and (NH₄)₂PtCl₄ to 3.5 Å resolution. A final mean figure of merit of 0.65 was obtained for 9628 reflections.With the aid of the amino acid sequence determined by Hogg &; Hermodson (1977), a complete model of the protein molecule has been determined using initially an optical comparator. The entire model was subsequently examined in detail using a computer graphic system.The protein molecule is ellipsoidal (axial ratio of 2:1), and consists of two globular domains (designated P and Q). Each domain is made from two separate polypeptide chain segments. Despite the discontinuity in the folding, the arrangements of the secondary structure in the two domains are very similar. Both domains contain a six-stranded parallel β-sheet (with the exception of the sixth anti-parallel strand in the Q domain) flanked by two α-helices on either side. The packing topology is α/β. A C-terminal helix is shared by both domains.The two domains show significant conformational similarity but lack sequence homology. A comparison of the two domains revealed that of the 139 α-carbons in the P domain and 152 in the Q domain, 92 were found to be equivalent with a root-mean-square distance of 2.6 Å.The cleft formed by the packing of the two domains is predominantly lined with hydrophilic residues. The sugar-binding site is located in this cleft.     

Molecular analysis of a gene fromBacteroides fragilis involved in metronidazole resistance inEscherichia coli

The region ofBacteroides fragilis DNA on the recombinant plasmid pMT100 responsible for conferring metronidazole resistance inEscherichia coli strains was characterized. An open reading frame (ORF1) of 195 bp encoded a protein of 64 amino acids with a predicted M_r, of 7.3 kDa. Deletion analysis indicated that ORF1 conferred the metronidazole resistance phenotype and encoded a protein with an apparent M_r of approximately 8–10 kDa.     

A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ

We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, P_REN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of P_REN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (M_r 30 006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).     

Characterizing the Escherichia coli O157:H7 proteome including protein associations with higher order assemblies

Background

The recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.

Methodology/Principal Findings

We examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate M_r ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high M_r fraction. Hundreds of proteins were enriched in a M_r range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster.

Significance

Nearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Two-dimensional lattices of porin diffract to 6 Å resolution

Porin (the product of gene ompF) is an integral membrane protein (M_r 36 500) of the outer membrane of Escherichia coli (strain B^E). The protein has been purified to homogeneity and reconstituted in dimyristoyl-lecithin. Oriented specimen on a flat surface yielded X-ray diffraction pattern, originating from the two-dimensional protein lattice, to a resolution reaching 6 Å. Although these powder rings are broad compared to corresponding diffraction patterns from purple membranes of Halobacterium halobium, porin is the first reconstituted integral membrane protein which shows diffraction to this resolution.     

Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of Streptococcus thermophilus

The lactose-H⁺ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIA^LacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIA^Glc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIA^LacS would be able to perform one or more functions associated with IIA^Glc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIA^LacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIA^LacS was unable to restore glucose uptake in a IIA^Glc-deficient strain, which is consistent with a very low rate of phosphorylation of IIA^LacS by phosphorylated HPr (HPr~P) from E. coli. With HPr~P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIA^LacS (k₁ = 4.3 × 10² M⁻¹ s⁻¹) and dephosphorylation of IIA^LacS~P by HPr (k₋₁ = 1.1 × 10³ M⁻¹ s⁻¹) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIA^Glc and HPr from E. coli. This finding suggests that IIA^LacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIA^Glc, we constructed a double mutant [IIA^LacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIA^LacS(I548E/G556D) by HPr~P increased (k₁ = 4.0 × 10³ M⁻¹ s⁻¹) and became nearly independent of the source of HPr~P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIA^LacS(I548E/G556D) was insufficient to complement IIA^Glc in PTS-mediated glucose transport in E. coli. Both IIA^LacS and IIA^LacS(I548E/G556D) could replace IIA^Glc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.