Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.     

Identification of the secY (prlA) gene product involved in protein export in Escherichia coli

Summary The gene secY (or prlA) is essential for protein export across the cytoplasmic membrane of Escherichia coli. The protein product of secY has been identified using the gene cloned under the control of the pL promoter of phage in combination with the maxicell system. The protein was found to have some unusual properties. First, it is important not to heat the protein at 100°C in the SDS sample buffer for its subsequent detection by gel electrophoresis. Second, migration of the protein in SDS-polyacrylamide gel electrophoresis is variable depending on the gel compositions. Gels with stronger sieving effect give higher apparent molecular weights. These properties are similar to those of hydrophobic proteins of the cytoplasmic membrane, such as the lactose permease. Finally, a major fraction of the protein synthesized from the overproducing plasmid is degraded rapidly in vivo. The altered protein from the secY24 mutant gene is even more unstable. These results provide information which is basic for the dissection of the protein export machinery of the bacterial cell.     

Analysis of a Coxiella burnetti gene product that activates capsule synthesis in Escherichia coli: requirement for the heat shock chaperone DnaK and the two-component regulator RcsC.

A 1.2-kb EcoRI genomic DNA fragment of Coxiella burnetti, when cloned onto a multicopy plasmid, was found to induce capsule synthesis (mucoidy) in Escherichia coli. Nucleotide sequence analysis revealed the presence of an open reading frame that could encode a protein of 270 amino acids. Insertion of a tet cassette into a unique NruI restriction site resulted in the loss of induction of mucoidy. Because of its ability to induce mucoidy, we designated this gene mucZ. Computer search for homologies to mucZ revealed 42% identity to an open reading frame located at 1 min of the E. coli chromosome. Interestingly, the C-terminal amino acid residues of MucZ share significant homology with the J domain of the DnaJ protein and its homologs, suggesting potential interactions between MucZ and components of the DnaK-chaperone machinery. Results presented in this paper suggest that E. coli requires DnaK-chaperone machinery for Lon-RcsA-mediated induction of capsule synthesis, as noticed first by S. Gottesman (personal communication). The induction caused by MucZ is independent of Lon-RcsA and is mediated through the two-component regulators RcsC and RcsB. DnaK and GrpE but not DnaJ are also required for the RcsB-mediated MucZ induction, and we propose that MucZ is a DnaJ-like chaperone protein that might be required for the formation of an active RcsA-RcsB complex and for the RcsC-dependent phosphorylation of RcsB. Discussions are presented that suggest three different roles for alternative forms of the DnaK-chaperone machinery in capsule production.     

secD, a new gene involved in protein export in Escherichia coli.

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.     

Continuous synthesis of the dnaA gene product of Escherichia coli in the cell cycle

Summary The dnaA gene product of Escherichia coli, identified as a weakly basic protein of about 48,000 daltons (Yuasa and Sakakibara 1980), can be separated from other celluar proteins by means of two-dimensional gel electrophoresis. Synthesis of the dnaA protein took place continuously during a cell growth cycle. The newly synthesized dnaA protein persisted stably for one generation. Thermosensitive dnaA protein produced by the dnaA167 mutant was stable at 30° C, but was disintegrated at 42° C. The amount of intact dnaA protein present in the mutant exposed to the high temperature for 60 min was less than a quarter of the amount at the time of the shift. The cells having the reduced amount of intact dnaA protein were capable of initiating a new round of chromosome replication at the low temperature without de novo synthesis of the dnaA protein. The potential of the mutant for initiation of DNA replication decreased with reduction in the amount of the thermoreversible dnaA protein. The mutations dnaA167 and dnaA46 had no significant effect on the syntheses of the dnaA mRNA and the protein product at the low and high temperatures.Abbreviations used SDS sodium dodecyl sulfate - kb kilobase pairs - TCA trichloroacetic acid     

Analysis of the relA gene product of Escherichia coli.

The relA gene product, ATP: GTP 3'-pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 degrees C of 120 mumol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 degrees C was found to be 4 mumol guanosine pentaphosphate formed min-1 mg protein-1. These values are about 100 times higher than reported by others. Our further studies of this enzyme led to the following results. Antibodies raised against this enzyme inhibit the ribosome-activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes. The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes. The amount of antibody-binding material in CP79 (relA) is at least 5 times lower.     

Identification of the purC gene product of Escherichia coli.

The purC region of the Escherichia coli chromosome was isolated from in vivo-derived lambda transducing bacteriophages and cloned in high-copy-number plasmids. The product of the purC gene, phosphoribosylaminoimidazolesuccinocarboxamide synthetase, was identified as a protein with an Mr of ca. 27,000. The level of the protein is increased by more than 60-fold in strains carrying the gene on a high-copy-number plasmid. Purine addition represses the enzyme level in both plasmid- and non-plasmid-containing strains.     

Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli

Escherichia coli was found to grow on fructoselysine as an energetic substrate at a rate of about one-third of that observed with glucose. Extracts of cells grown on fructoselysine catalyzed in the presence of ATP the phosphorylation of fructoselysine and a delayed formation of glucose 6-phosphate from this substrate. Data base searches allowed us to identify an operon containing a putative kinase (YhfQ) belonging to the PfkB/ ribokinase family, a putative deglycase (YhfN), homologous to the isomerase domain of glucosamine-6-phosphate synthase, and a putative cationic amino acid transporter (YhfM). The proteins encoded by YhfQ and YhfN were overexpressed in E. coli, purified, and shown to catalyze the ATP-dependent phosphorylation of fructoselysine to a product identified as fructoselysine 6-phosphate by 31P NMR (YhfQ), and the reversible conversion of fructoselysine 6-phosphate and water to lysine and glucose 6-phosphate (YhfN). The K(m) of the kinase for fructoselysine amounted to 18 microm, and the K(m) of the deglycase for fructoselysine 6-phosphate, to 0.4 mm. A value of 0.15 m was found for the equilibrium constant of the deglycase reaction. The kinase and the deglycase were both induced when E. coli was grown on fructoselysine and then reached activities sufficient to account for the rate of fructoselysine utilization.     

Proton-motive-force-dependent step in the pathway to lysis of Escherichia coli induced by bacteriophage phi X174 gene E product.

We examined the cellular effects after the expression of the cloned lysis gene E of bacteriophage phi X174. Chloramphenicol prevented lysis only when added within the first minute of derepression of E synthesis, indicating that a time lag of several minutes exists between the synthesis of the E protein and the onset of cell lysis. Experiments with protonophores showed the existence of a subsequent step dependent on proton motive force at about 3 to 5 min before lysis.     

Role of the hemA gene product and delta-aminolevulinic acid in regulation of Escherichia coli heme synthesis.

We initiated these studies to help clarify the roles of heme, delta-aminolevulinic acid (ALA), hemA, and hemM in Escherichia coli heme synthesis. Using recombinant human hemoglobin (rHb1.1) as a tool for increasing E. coli's heme requirements, we demonstrated that heme is a feedback inhibitor of heme synthesis. Cooverexpression of rHb1.1 and the hemA-encoded glutamyl-tRNA (GTR) reductase increased intracellular levels of ALA and heme and increased the rate of rHb1.1 formation. These results support the conclusion that heme synthesis is limited by ALA (S. Hino and A. Ishida, Enzyme 16:42-49, 1973; W. K. Philipp-Dormston and M. Doss, Enzyme 16:57-64, 1973) and that the hemA-encoded GTR reductase is a rate-limiting enzyme in the pathway (J.-M. Li, C. S. Russell, and S. D. Cosloy, Gene 82:2099-217, 1989). Increasing the copy number of hemM, whose product is believed to be required for efficient ALA formation (W. Chen, C. S. Russell, Y. Murooka, and S. D. Cosloy, J. Bacteriol. 176:2743-2746, 1994; M. Ikemi, K. Murakami, M. Hashimoto, and Y. Murooka, Gene 121:127-132, 1992), had no effect on either ALA pools or the rate of rHb1.1 accumulation. The hemA-encoded GTR reductase was found to be regulated by ALA. Some of our results differ from those reported by Hart and coworkers (R. A. Hart, P. T. Kallio, and J. E. Bailey, Appl. Environ. Microbiol. 60:2431-2437, 1994), who concluded that ALA formation is not the rate-limiting step in E. coli cells expressing Vitreoscilla hemoglobin.     

An in vitro complementation assay for the Escherichia coli uvrD gene product.

An in vitro assay specific for the product of the uvrD gene of Escherichia coli has been developed. This assay, derived from properties of uvrD mutants revealed by in vivo experiments, is based on the necessity for a functional UvrD protein for complete rejoining of covalently closed circular DNA during the excision repair of UV-induced damage. Extracts prepared from gently lysed uvrD101 mutant cells are capable of restoring UV-damaged DNA to its covalently closed circular form when provided with a functional UvrD protein from other repair-deficient cell extracts or from partially purified protein fractions. This assay was employed to monitor the activity of the UvrD protein after several steps of fractionation. The partially purified UvrD protein does not complement extracts deficient in DNA polymerase I or temperature-sensitive in DNA ligase; it does, however, complement extracts from strains mutant at the uvrE and recL loci, which are considered allelic with the uvrD locus.     

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.     

The deoxycytidine pathway for thymidylate synthesis in Escherichia coli

When thymidylate production is diminished by a mutation affecting dCTP deaminase, Escherichia coli is known to use an alternate pathway involving deoxycytidine as an intermediate. The pathway requires the gene for any of three nucleoside diphosphate kinases (ndk, pykA, or pykF) and the gene for a 5′-nucleotidase (yfbR).     

Regulation of the Escherichia coli glyA gene by the purR gene product.

The purine regulon repressor protein, PurR, was shown to be a purine component involved in glyA regulation in Escherichia coli. Expression of glyA, encoding serine hydroxymethyltransferase activity, was elevated in a purR mutant compared with a wild-type strain. When the purR mutant was transformed with a plasmid carrying the purR gene, the serine hydroxymethyltransferase levels returned to the wild-type level. The PurR protein bound specifically to a DNA fragment carrying the glyA control region, as determined by gel retardation. In a DNase I protection assay, a 24-base-pair region was protected from DNase I digestion by PurR. The glyA operator sequence for PurR binding is similar to that reported for several pur regulon genes.     

Identification of the Escherichia coli cya gene product as authentic adenylate cyclase

The Escherichia coli cya gene has been fused in the same register with the lacZ gene. The corresponding hybrid cya-lacZ gene is expressed as a bifunctional protein that exhibits both adenylate cyclase and beta-galactosidase activities, thus proving that cya is the structural gene for adenylate cyclase. The hybrid protein was purified to homogeneity and has been used to raise antibodies that recognize wild-type adenylate cyclase. Finally, the protein has been submitted to amino acid sequence analysis. It has been found that the first ten amino acids fit the predicted sequence obtained from DNA sequence analysis, thus substantiating the prediction that the cya translation initiation codon is UUG .     

Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells.

Saccharomyces cerevisiae MATa cells release a lipopeptide mating pheromone, a-factor. Radiolabeling and immunoprecipitation show that MATa ste6 mutants produce pro-a-factor and mature a-factor intracellularly, but little or no extracellular pheromone. Normal MATa cells carrying a multicopy plasmid containing both MFa1 (pro-a-factor structural gene) and the STE6 gene secrete a-factor at least five times faster than the same cells carrying only MFa1 in the same vector. The nucleotide sequence of the STE6 gene predicts a 1290 residue polypeptide with multiple membrane spanning segments and two hydrophilic domains, each strikingly homologous to a set of well-characterized prokaryotic permeases (including hlyB, oppD, hisP, malK and pstB) and sharing even greater identity with mammalian mdr (multiple drug resistance) transporters. These results suggest that the STE6 protein in yeast, and possibly mdr in animals, is a transmembrane translocator that exports polypeptides by a route independent of the classical secretory pathway.     

The requirement for energy during export of beta-lactamase in Escherichia coli is fulfilled by the total protonmotive force.

The energy requirement for the maturation and export of the plasmid-encoded TEM beta-lactamase in Escherichia coli K12 was shown to be fulfilled by the total protonmotive force. This was demonstrated by assessing the inhibition of proteolytic processing of the precursor form of beta-lactamase caused by perturbation of the energized state of the membrane in cells treated with valinomycin. The magnitude of the membrane potential was manipulated by varying the concentration of KCl in the medium and the pH gradient was manipulated by varying the external pH. Both components were simultaneously affected by addition of the protonophore carbonylcyanide-p- trifluoromethoxy phenylhydrazone (FCCP). Inhibition of processing was demonstrated in a mutant strain having a defective ATP synthase where protonmotive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition was not the result of decreased ATP concentration. Half-maximal accumulation of precursor of beta-lactamase was observed in all cases when the level of protonmotive force was decreased to approximately 150 mV. Under those conditions the membrane potential varied from 65 to 140 mV (internally negative) and the pH gradient from 95 to 25 mV (internally alkaline). Thus, the energy requirement is satisfied by the total protonmotive force, with no specificity for either the membrane potential or the pH gradient.     

High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli

Numerous high‐value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin‐arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed‐batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial‐type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA‐GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co‐expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over‐expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed‐batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h^?1, the cultures reached OD₆₀₀ values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L^?1 culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec‐dependent export approaches. Biotechnol. Bioeng. 2012; 109: 2533–2542. © 2012 Wiley Periodicals, Inc.     

Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance.

There are DnaA protein-binding sites in at least one F origin of replication, and only potentially leaky dnaA(Ts) mutations had ever been used in previous studies indicating that F replication was independent of the dnaA gene product. Here we show that an Escherichia coli dnaA::Tn10 host which does not make a dnaA gene product cannot sustain autonomous or integrated F plasmid maintenance.